11β-Amidoalkyl estradiols, a new series of pure antiestrogens

In order to find new antiestrogens, devoid of any agonistic activity, a series of 11β-amidoalkyl estradiols were prepared. These compounds have been studied in comparison with tamoxifen (TAM): in vitro, for their relative binding affinities (RBA) for mouse and MCF-7 estrogen receptors (ER) and for their antiproliferative effect on MCF-7 (estradiol or EGF/PDGF stimulated) and Ly2 human breast cancer cell lines; in vivo, for their uterotrophic/antiuterotrophic activities in the mouse and for their antitumoral activities on MCF-7 tumors implanted in nude mice.

The most representative compounds are N-methyl-N-isopropyl-(3,17β-dihydroxy-estra-1,3,5(10)-trien-11β-yl)-undecanamide (RU 51625) and its 17-ethynyl derivative (RU 53637). They showed good RBAs for ER and a stronger antiproliferative effect than TAM in vitro. Unlike TAM, these compounds inhibited growth factor stimulated MCF-7 proliferation, and the growth of the TAM resistant cell line Ly2. In vivo, they were completely devoid of uterotrophic activity, when given subcutaneously in mice, but exhibited a slight agonistic effect when administered orally. They showed interesting antitumor activities in nude mice by the percutaneous route, but RU 53637 was significantly more potent than RU 51625 when given orally.     

The metabolism of dihydrotachysterols: Renal side chain and non-renal nuclear hydroxylations in vivo and in vitro

The metabolism of dihydrotachysterol (DHT), a hydrogenated analogue of vitamin D, has been studied in vivo using man and rat and in vitro using the perfused rat kidney, and hepatoma (3B) and osteosarcoma (UMR-106) cell lines. In vivo a large number of metabolites appeared in the plasma of rats given DHT₂ and DHT₃. Of particular interest was a compound more polar than 25-hydroxy-DHT, which has been designated compound H. Further study of this compound showed that it was composed of two components, one (Ha) being in much lower concentration than the other (Hb). The production of T2/H (peak H from DHT₂) was demonstrated in human plasma after administration of oral DHT₂. Comparison of the metabolites formed in vivo with those isolated from the rat kidney perfused with 25-hydroxy-DHT₃ in vitro showed that 25-hydroxy-DHT₃ was metabolized along two metabolic pathways previously described for vitamin D, culminating in the production of 25-hydroxy-DHT₃-23,26-lactone and 23,25-dihydroxy-24-oxo-DHT₃. The osteosarcoma cell line metabolized 25-OH-DHT₃ in vitro along the same two metabolic pathways already demonstrated in the perfused rat kidney. More polar metabolites than compound H seen in rat plasma in vivo were shown to be metabolites of compound H and similar metabolites were also produced in the osteosarcoma cell line from chemically synthesized 1,25-dihydroxy-DHT₃. The hepatoma cell line 25-hydroxylated DHT and no feed-back inhibition was observed. Use of the hepatoma cell to 25-hydroxylate a number of chemically synthesized 1-hydroxy-DHTs indicated that compound Ha was indistinguishable from 1,25-dihydroxy-DHT whereas compound Hb is possibly 1β,25-dihydroxy-DHT. Studies with the VDR in both chick gut and calf thymus indicated that 1,25-dihydroxy-DHT is very effective in displacing radiolabelled 1,25-dihydroxyvitamin-D₃ and is thus most likely to be the calcaemic metabolite of DHT.     

A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro

Exposure of endothelium to a nominally uniform flow field in vivo and in vitrofrequently results in a heterogeneous distribution of individual cell responses. Extremes in response levels are often noted in neighboring cells. Such variations are important for the spatial interpretation of vascular responses to flow and for an understanding of mechanotransduction mechanisms at the level of single cells. We propose that variations of local forces defined by the cell surface geometry contribute to these differences. Atomic force microscopy measurements of cell surface topography in living endothelium both in vitro and in situ combined with computational fluid dynamics demonstrated large cell-to-cell variations in the distribution of flow-generated shear stresses at the endothelial luminal surface. The distribution of forces throughout the surface of individual cells of the monolayer was also found to vary considerably and to be defined by the surface geometry. We conclude that the endothelial three-dimensional surface geometry defines the detailed distribution of shear stresses and gradients at the single cell level, and that there are large variations in force magnitude and distribution between neighboring cells. The measurements support a topographic basis for differential endothelial responses to flow observed in vivo and in vitro. Included in these studies are the first preliminary measurements of the living endothelial cell surface in an intact artery.     

Effects of progestagens and Org OD14 in in vitro and in vivo tumor models

Sex steroids, in particular estradiol (E2) and progesterone (P4), play, together with other hormones and growth factors, a role in the development of normal breast tissue. The effect of four progestagens (norethisterone, 3-ketodesogestrel, gestodene and P4) and Org OD14, a steroid with weak estrogenic, progestagenic and androgenic properties were studied on growth of breast tumor cells in vitro using two subclones of MCF-7 (H and A) and T47D (S and A) cells. In addition, we investigated the effects of 3-ketodesogestrel, gestodene and Org OD14 on the growth of 7,12-dimethyl-benz(a)anthracene(DMBA)-induced mammary tumors in rats. In the in vitro assays with MCF-7 cells norethisterone, 3-ketodesogestrel and gestodene stimulated growth only at high doses (10⁻⁷ M), whereas P4 had no effect. Gestodene was more potent than 3-ketodesogestrel and norethisterone. Org OD14, stimulated cell growth at a dose of 10⁻⁸ M, while E2 is active at 10⁻¹⁰ M. In T47D-A cells similar effects were found, but the subclone S did not respond to the progestagens and Org OD14. The two T47D subclones also reacted differently to progestagens during growth stimulation with E2. In T47D-S the progestagens and Org OD14 inhibited, while in T47D-A these compounds did not modulate the effect of E2. In the DMBA model we found that gestodene and 3-ketodesogestrel were able to inhibit tumor growth to the same extent. Surprisingly, Org OD14 was even more effective in the DMBA model using the therapeutic approach. Using the prophylaxic approach tumor development was delayed and tumor growth was strongly suppressed. The inhibitory effects of Org OD14 on tumor growth in the DMBA model may be attributed to its mixed hormonal profile. From these studies we conclude that different cell lines and even subclones thereof respond quite differently to steroids. Both in vitro and in vivo studies are required to judge whether synthetic steroids might be involved in an increased risk for the development of breast tumors.     

Pasteur effect in the in vitro vascularly perfused rat small intestine

Isolated small intestine perfused in vitro with media with low oxygen concentration was found to contain low levels of ATP when compared with rat small intestine in vivo. The addition of fluorocarbon FC 75 to an erythrocyte-free perfusion medium was found to result in a high phosphate potential and a low rate of lactate production from glucose in isolated perfused small intestine, resembling the in vivo condition. This allowed the demonstration of a Pasteur effect in that replacement of oxygen by nitrogen (or the addition of 2,4-dinitrophenol) led to a rapid increase of the rate of glycolysis, and a decrease of the ATP concentration in the tissue     

In vitro and in vivo interactions between nuclear receptors at estrogen response elements

To study mechanisms involved in the antiestrogenic effect of retinoic acid (RA), previously described in mammalian cells, we used in vitro and in vivo approaches. One hypothesis was direct competition between nuclear receptors (ER, RAR and RXR) at the DNA level. We first showed in vitro that the RAR/RXR heterodimer could weakly bind an ERE and that retinoid receptors reduced binding of ER to an ERE. We next checked whether, in yeast, direct competition between receptors that recognize the same responsive element could be monitored in a reconstituted heterologous estrogen-responsive system, by determining the expression of a reporter gene. We then co-transformed RAR and RXR in an estrogenic responsive strain. This model demonstrated that, even though RAR/RXR was able to bind an ERE, the addition of retinoic acid had no inhibitory effect on estrogen-induced responses in this yeast system, unlike in mammalian cells. Interference between these receptors should require other factors than interactions at the ERE level. This model could be used to identify mammalian factors interacting with estrogen and retinoic acid receptors which could play a role in crosstalk between these receptors.     

Tissue selective action of tamoxifen methiodide, raloxifene and tamoxifen on creatine kinase B activity in vitro and in vivo

We have compared the cell and tissue selective estrogenic and antiestrogenic activities of tamoxifen, raloxifene, ICI 164,384 and a permanently ionized derivative of tamoxifen — tamoxifen methiodide (TMI). This non-steroidal antiestrogen has limited ability to cross the blood brain barrier and is therefore less likely to cause the central nervous system disturbances caused by tamoxifen. We have used the stimulation of the specific activity of the “estrogen induced protein”, creatine kinase BB, as a response marker in bone, cartilage, uterine and adipose cells and in rat skeletal tissues, uterus and mesometrial adipose tissue. In vitro, TMI, tamoxifen and raloxifene mimicked the agonistic action of 17β-estradiol in ROS 17/2.8 rat osteogenic osteosarcoma, female calvaria, and SaOS₂ human osteoblast cells. In Ishikawa endometrial cancer cells, tamoxifen showed reduced agonistic effects and raloxifene showed no stimulation. However, as antagonists, tamoxifen and raloxifene were equally effective in Ishikawa or SaOS₂ cells. In immature rats, all four of the antiestrogens inhibited estrogen action in diaphysis, epiphysis, uterus and mesometrial adipose tissue; when administered alone, tamoxifen stimulated creatine kinase (CK) specific activity in all these tissues. Raloxifene and TMI, however, stimulated only the skeletal tissues and had no stimulatory effect in the uterus or mesometrial fat, and the pure antiestrogen ICI 164,384 showed no stimulatory effect in any of the tissues. The simultaneous injection of estrogen, plus an antiestrogen which acted as an agonist, resulted in lower CK activity than after injection of either agent alone. These differential effects, in vivo and in vitro, may point the way to a wider therapeutic choice of an appropriate antiestrogen which, although antagonizing E₂ action in mammary cancer, can still protect against osteoporosis and cardiovascular disease and not stimulate the uterus with its attendant undesirable changes, or interfere with the beneficial action of E₂ in the brain.     

The blood-brain barrier in vitro: The second decade

Ever since the discovery of Paul Ehrlich (1885 Das Sauerstoff-bedürfnis des Organismus: Hirschwald, Berlin) about the restricted material exchange, existing between the blood and the brain, the ultimate goal of subsequent studies has been mainly directed towards the elucidation of relative importance of different cellular compartments in the peculiar penetration barrier consisting the structural basis of the blood-brain barrier (BBB). It is now generally agreed that, in most vertebrates, the endothelial cells of the central nervous system (CNS) are responsible for the unique penetration barrier, which restricts the free passage of nutrients, hormones, immunologically relevant molecules and drugs to the brain. After an era of studying with endogenous or exogenous tracers the unique permeability properties of cerebral endothelial cells in vivo, the next generation, i.e. the in vitro blood-brain barrier model system was introduced in 1973. Recent advances in our knowledge of the BBB have in part been made by studying the properties and function of cerebral endothelial cells (CEC) with this in vitro approach. This review summarizes the results obtained on isolated brain microvessels in the second decade of its advent.     

In vivo strain gage implantation in rats

A technique for the fabrication of encapsulated micro-miniature rosette strain gages for in vivo implantation is described. The gage units have an overall area of ten square millimeters (2.5 mm × 4.0 mm), and hence can be installed in very small experimental animals, particularly rodents. Using a rat model, strain data for up to 12 days have been obtained and in vitro studies have validified the in vivo strain recordings.     

Improved anti-tumoral capacity of mixed and pure anti-oestrogens in breast cancer cell xenografts after their administration by entrapment in colloidal nanosystems

Anti-oestrogens (AEs) are currently used for treating hormone-dependent breast cancers. They specifically bind to oestrogen receptors (ERs) and inhibit their transactivation capacity. However, ERs are present in various other tissues in which AEs may have either a beneficial or detrimental action. AE administration via systems targeting breast tumours may be an important therapeutic improvement. Thus, several biodegradable drug delivery systems containing either “mixed” (4-hydroxytamoxifen - 4-HT) or “pure” (RU 58668 - RU) AEs were prepared. Liposomes and nanospheres (NS, composed of non-toxic and biodegradable lipids and poly(d,l-lactic acid) incorporated up to 1 and 0.5 mM AE, respectively. Nanocapsules (NCs) in which an oily core solubilises the AE incorporated no more than 0.02 mM of the drug. PEG-functionalised nanoparticles survived longer in plasma and had better controlled release of the drug. The small size of the vectors (100–250 nm) was compatible with their extravasation through the discontinuous endothelium of tumour vasculature, allowing their accumulation in MCF-7 cell xenografts and leading to a prolonged exposure of the tumour to AEs. In these tumours and in MCF-7/ras xenografts, RU-NS and RU-NC (6.5 mg/kg/week and 0.27 mg/kg/week, respectively, doses at which free RU had a very weak effect), both inhibited tumour growth. Entrapped RU significantly induced involution of tumours and strongly induced apoptosis in tumour cells, concomitantly with inhibiting tumour angiogenesis. 4-HT-nanoparticles also arrest oestradiol-induced tumour growth, inducing apoptosis and inhibiting angiogenesis. However, unlike RU-nanoparticles, they did not promote ER subtype loss in tumour cells. Subcutaneous administration of both RU- and 4-HT-NS in MCF-7 xenografts strongly arrested tumour growth for prolonged periods and RUNS decreased the number of tumour epithelial cells. Analysis of the proteins involved in cell cycle proliferation and apoptosis confirmed that RU-nanoparticles were more efficient than 4-HT-nanoparticles. Their lack of toxicity and high anti-tumour potency that affects only tumour cells in the xenograft models mean these AE-loaded colloidal systems are a breakthrough in hormone-dependent breast cancer treatment.     

The effects of in vivo and in vitro non-enzymatic glycosylation and glycoxidation on physico-chemical properties of haemoglobin in control and diabetic patients

The erythrocyte deformability, which is related to erythrocyte internal viscosity, was suggested to depend upon the physico-chemical properties of haemoglobin. In the present study we employed ESR spectroscopy in order to explore further the extent to which the in vivo or in vitro glycation and/or glycoxidation might affect haemoglobin structure and conformation. We revealed that under both in vivo and in vitro conditions the attachment of glucose induced a mobilization of thiol groups in the selected domains of haemoglobin molecules (the increased h₊₁/h₀ parameter of maleimide spin label, MSL; 0.377 ± 0.021 in diabetics vs 0.338 ± 0.017 in controls, n = 12, P < 0.0001). The relative rotational correlation time (τ_c) of two spin labels, TEMPONE and TEMPAMINE, respectively, in erythrocyte insides (5.22 ± 0.42 in diabetics, n = 21 vs 4.79 ± 0.38, n = 16 in controls, P < 0.005) and in the solutions of in vitro glycated haemoglobin, were increased. Neither oxidation nor crosslinking of thiol groups was evidenced in glycated and/or oxidized haemoglobin. In addition, erythrocyte deformability was found to be reduced in type 2 diabetic patients (6.71 ± 1.08, n = 28 vs 7.31 ± 0.96, n = 21, P < 0.015). In conclusion, these observations suggest that: the attachment of glucose to haemoglobin might have decreased the mobility of the Lys-adjacent Cys residues, thus leading to the increased h₊₁/h₀ parameter of MSL. Such structural changes in haemoglobin owing to non-enzymatic glycosylation may contribute to the increased viscosity of haemoglobin solutions (r = 0.497, P < 0.0035) and the enhanced internal viscosity of diabetic erythrocytes (r = 0.503, P < 0.003). We argue that such changes in haemoglobin, and consequently in red blood cells, might contribute to the handicapped oxygen release under tissue hypoxia in the diabetic state.     

Translating tissue culture results into animal models: the case of Salmonella typhimurium

Investigators use both in vitro and in vivo models to better understand infectious disease processes. Both models are extremely useful in research, but there exists a significant gap in complexity between the highly controlled reductionist in vitro systems and the largely undefined, but relevant variability encompassing in vivo animal models. In an effort to understand how Salmonella initiates disease at the intestinal epithelium, in vitro models have served a useful purpose in allowing investigators to identify molecular mechanisms responsible for Salmonella invasion of host cells and stimulation of host inflammatory responses. Identification of these molecular mechanisms has generated hypotheses that are now being tested using in vivo models. Translating the in vitro findings into the context of an animal model and subsequently to human disease remains a difficult challenge for any disease process.     

Are breast tumours resistant to tamoxifen also resistant to pure antioestrogens?

A substantial proportion of patients with breast cancer are treated with the antioestrogen tamoxifen. As with other endocrine therapies, clinical experience has shown that some tumours in which growth is initially attenuated by tamoxifen treatment become resistant to continued drug treatment and resume growth. The mechanisms underlying the development of tamoxifen resistance have yet to be described but represent an important focus of research with the aim of defining what other therapies might be effective following tamoxifen treatment. Secondly, an understanding of tamoxifen resistance might suggest means to develop more effective agents for primary treatment of the disease. The development of pure antioestrogens, for example ICI 164,384 and ICI 182,780, which differ pharmacologically from tamoxifen in being entirely free of oestrogen partial-agonist activity, together with cell and animal models of tamoxifen resistant human breast cancer, has revealed one mechanism which might be of considerable clinical significance. Pure antioestrogens were shown to inhibit the proliferation of a greater proportion of tumor cells than tamoxifen in vitro, a differential effect that was attributed to the oestrogenic activity of tamoxifen. Subsequently, cell culture studies have shown that breast cancer cell lines selected for resistance to tamoxifen can still remain sensitive to the growth inhibitory action of pure antioestrogens. Similarly, the growth of human breast tumours in nude mice, which is initially attenuated by tamoxifen but then resumes, can be inhibited by pure antioestrogens. Both types of experiment are consistent with the view that tamoxifen resistance in these model systems is due to the oestrogenic action of tamoxifen. Thus, it can be predicted that in some patients whose tumours recur during tamoxifen therapy, a further response to pure antioestrogen treatment might occur. Studies to examine this hypothesis are currently being undertaken with ICI 182,780. One mechanism which might account for the experimental observations is an intrinsic heterogeneity amongst breast tumour cells in their response to tamoxifen, i.e. that there are at least two different populations of cells; one population which responds to tamoxifen as an antioestrogen and one which “reads” tamoxifen as an oestrogen. The growth advantage thus conferred on the latter population would lead to its predominance. If this is what actually happens in a proportion of human tumours, it can be argued that primary treatment of the tumour with a pure antioestrogen, rather than tamoxifen, would be preferred since a more complete and longer-lasting response would be predicted. Recent comparative studies with human breast tumours grown in nude mice support these predictions.     

Early markers of in vitro microspore reprogramming to embryogenesis in olive (Olea europaea L.)

Microspore embryogenesis to form haploid and double-haploid embryos and regenerated plants is an efficient method of producing homozygous lines for crop breeding. In trees, the process is of special interest since classical methods are impractical in many cases, as in Olea europaea L. Recently, a convenient method has been developed for microspore embryogenesis induction by stress in olive isolated microspores in vitro cultures. In the present work, the switch of the microspore developmental pathway and the formation of microspore-derived multicellular proembryos have been achieved and a cytochemical and immunocytochemical analysis was performed in the early stages. The young microspore proembryos displayed defined features different to both, the in vivo gametophytic, and the in vitro non-responsive microspores. Reprogrammed microspores showed an absence of starch, the occurrence of a first symmetrical division and cytokinesis, the presence of an abundant ribosomal population, and changes in cellulosic and pectic cell wall components which constituted early markers of the embryogenic microspore process. They provided new insights on the molecular and cellular events associated with the microspore reprogramming of woody plants, and specifically in olive, providing interesting knowledge which could guide future selection and regeneration strategies in this fruit tree of high economic interest.     

Specific effect of manganese on the allantoinase of Vigna Radiata

Vigna radiata seedlings germinated in the presence of Mn²⁺ show an unusual increase in allantoinase activity which is proportional to Mn²⁺ concentration up to 5 mM. Though Mn²⁺ is not an activator for V. radiata allantoinase, it specifically protects allantoinase against thermal as well as papain-catalysed inactivation. Evidence is presented to show that the primary effect of Mn²⁺ is a protective one, both in vitro and in vivo, and that this is reflected in the observed enhancement of allantoinase activity in Mn²⁺ grown seedlings. That this unusual effect of Mn²⁺ is a specific one is indicated by the lack of a similar effect with Mg²⁺. Cu²⁺ is shown to destabilize V. radiata allantoinase in vitro as well as in vivo.     

Bovine babesiosis: Partial purification and characterization of blood culture-derived Babesia bovis

Morphologic, biologic and immunologic properties of corpuscular and soluble fractions of Babesia bovis purified from in vitro blood cultures were studied. Supernatant fluids obtained during routine medium exchange were studied. Supernatant fluids obtained during routine medium exchange were submitted to differential centrifugation to separate soluble and corpuscular babesial antigens from erythrocyte stroma. Extracellular babesiae were sedimented with infected and uninfected erythrocyte ghosts. The majority of babesiae were found in erythrocyte ghosts. Clumps of extracellular parasites were sometimes formed in vitro and generally could not be separated from uninfected erythrocytes. Centrifugation over a discontinuous Ficoll density gradient did not improve separations. Parasites remained viable throughout the purification procedure but were killed by freezing and rapid thawing. Both corpuscular and soluble antigen fractions elicited the production of specific anti-babesial antibodies when injected into calves. Electron microscopy of corpuscular antigen revealed the presence of intra- and extraerythrocytic babesial merozoites. A surface coat was visible loosely adhering to the plasma membrane of the parasites. Parasite suspensions and cell-free supernatant fluids obtained from in vitro cultures of B. bovis should provide a variety of unique antigens for further in vitro and in vivo studies.     

Developmental profiles of glial enzymes in the chick embryo: In vivo and in culture

The activities of two glial cell enzymes, glutamine synthetase (a marker for astrocytes) and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (a marker for oligodendrocytes and myelination) were studied in the developing chick embryo brain in vivo and in cultures derived from chick embryos. The in vivo findings showed that the activities of both enzymes parallel the patterns of gliogenesis and myelination. Glutamine synthetase follows similar patterns in culture and in vivo, whereas the developmental profile of 2′,3′-cyclic nucleotide 3′-phosphohydrolase appears to be affected by the culture conditions.     

Corticotropin releasing factor decidualizes human endometrial stromal cells in vitro. Interaction with progestin

Decidualization of endometrial cells is a hormone-dependent process of differentiation which occurs during the menstrual cycle and pregnancy. Recent in vitro studies have revealed that cAMP and its generators induce decidualization of stromal cells isolated from proliferative endometrium and that progestins enhance the effect of cAMP. Since corticotropin releasing factor (CRF) generates cAMP and prostaglandins in other organs, in the present study the effect of CRF, a hypothalamic factor also produced by decidua and fetal membranes, on in vitro decidualization of endometrial stromal cells was evaluated. The addition of CRF to a culture medium of stromal cells induced in vitro decidualization, as indicated by morphologic changes from elongated fibroblast-like cells into larger and round cells and by the release of prolactin in the medium. The effect of CRF on stromal cells and on prolactin release was significantly augmented by the coincubation in the presence of medroxyprogesterone acetate. This observation indicates CRF as a novel factor of decidualization and confirms that progestins act as enhancers of the expression of decidual products.