BRCA1 and GATA3 corepress FOXC1 to inhibit the pathogenesis of basal-like breast cancers

In this study we describe a novel interaction between the breast/ovarian tumor suppressor gene BRCA1 and the transcription factor GATA3, an interaction, which is important for normal breast differentiation. We show that the BRCA1-GATA3 interaction is important for the repression of genes associated with triple-negative and basal-like breast cancer (BLBCs) including FOXC1, and that GATA3 interacts with a C-terminal region of BRCA1. We demonstrate that FOXC1 is an essential survival factor maintaining the proliferation of BLBCs cell lines. We define the mechanistic basis of this corepression and identify the GATA3-binding site within the FOXC1 distal promoter region. We show that BRCA1 and GATA3 interact on the FOXC1 promoter and that BRCA1 requires GATA3 for recruitment to this region. This interaction requires fully functional BRCA1 as a mutant BRCA1 protein is unable to localize to the FOXC1 promoter or repress FOXC1 expression. We demonstrate that this BRCA1-GATA3 repression complex is not a FOXC1-specific phenomenon as a number of other genes associated with BLBCs such as FOXC2, CXCL1 and p-cadherin were also repressed in a similar manner. Finally, we demonstrate the importance of our findings by showing that loss of GATA3 expression or aberrant FOXC1 expression contributes to the drug resistance and epithelial-to-mesenchymal transition-like phenotypes associated with aggressive BLBCs. Oncogene (2012) 31, 3667-3678; doi:10.1038/onc.2011.531; published online 28 November 2011

Rheological, mechanical and membrane penetration properties of novel dual drug systems for percutaneous delivery

In this study it has been demonstrated that mixtures of two solid drugs, ibuprofen and methyl nicotinate, with different but complementary pharmacological activities and which exist as a single liquid phase over a wide composition range at skin temperature, can be formulated as o/w emulsions without the use of an additional hydrophobic carrier. These novel dual drug systems provided significantly enhanced in vitro penetration rates through a model lipophilic barrier membrane compared to conventional individual formulations of each active. Thus, for ibuprofen, drug penetration flux enhancements of three- and 10-fold were observed when compared to an aqueous ibuprofen suspension and a commercial alcohol-based ibuprofen formulation, respectively. Methyl nicotinate penetration rates were shown to be similar for aqueous gels and emulsified systems. Mechanisms explaining these observations are proposed. Novel dual drug formulations of ibuprofen and methyl nicotinate, formulated within the liquid range at skin temperature, were investigated by oscillatory rheology and texture profile analysis. demonstrating the effects of drug and viscosity enhancer concentrations, and disperse phase type upon the rheological, mechanical and drug penetration properties of these systems. (C) 2000 Elsevier Science B.V. All rights reserved.

Feedback via Ca²⁺-activated ion channels modulates endothelin 1 signaling in retinal arteriolar smooth muscle

PURPOSE: To investigate the role of feedback by Ca²?-sensitive plasma-membrane ion channels in endothelin 1 (Et1) signaling in vitro and in vivo. Methods. Et1 responses were imaged from Fluo-4-loaded smooth muscle in isolated segments of rat retinal arteriole using two-dimensional (2-D) confocal laser microscopy. Vasoconstrictor responses to intravitreal injections of Et1 were recorded in the absence and presence of appropriate ion channel blockers using fluorescein angiograms imaged using a confocal scanning laser ophthalmoscope. Results. Et1 (10 nM) increased both basal [Ca²?](i) and the amplitude and frequency of Ca²?-waves in retinal arterioles. The Ca²?-activated Cl?-channel blockers DIDS and 9-anthracene carboxylic acid (9AC) blocked Et1-induced increases in wave frequency, and 9AC also inhibited the increase in amplitude. Iberiotoxin, an inhibitor of large conductance (BK) Ca²?-activated K?-channels, increased wave amplitude in the presence of Et1 but had no effect on frequency. None of these drugs affected basal [Ca²?](i). The voltage-operated Ca²?-channel inhibitor nimodipine inhibited wave frequency and amplitude and also lowered basal [Ca²?](i) in the presence of Et1. Intravitreal injection of Et1 caused retinal arteriolar vasoconstriction. This was inhibited by DIDS but not by iberiotoxin or penitrem A, another BK-channel inhibitor. Conclusions. Et1 evokes increases in the frequency of arteriolar Ca²?-waves in vitro, resulting in vasoconstriction in vivo. These responses, initiated by release of stored Ca²?, also require positive feedback via Ca²?-activated Cl?-channels and L-type Ca²?-channels.

Path-selective photoinduced electron transfer (PET) in a membrane-associated system studied by pH-dependent fluorescence

The pH-dependent fluorescence behavior of two regioisomeric 'receptor(1)-spacer(1)-fluorophore-spacer(2)-receptor(2)' systems 1 and 2 in micellar solutions of sodium dodecyl sulfate show that photoinduced electron transfer (PET) only occurs from the amine group connected to the 4-amino position of the aminonaphthalimide fluorophore in both cases. This demonstrates the directing influence of the photogenerated electric field within the aminonaphthalimide excited state on the electron transfer process. Since path-selectivity of PET is also known within the membrane-bound photosynthetic reaction center in bacteria, its origins may be illuminated by the simple experiments described here. (C) 2011 Elsevier B. V. All rights reserved.

FASCIOLA-HEPATICA - ULTRASTRUCTURAL-CHANGES TO THE TEGUMENT OF JUVENILE FLUKES FOLLOWING INCUBATION IN-VITRO WITH THE DEACETYLATED (AMINE) METABOLITE OF DIAMPHENETHIDE

Ultrastructural changes to the tegument of 5-week-old, 3-week-old and freshly-excysted Fasciola hepatica following in vitro incubation with the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 mu gml(-1)) were examined by transmission electron microscopy, A similar sequence of tegumental changes occurred in all three age groups of fluke, although, with increasing fluke age, the time before onset increased and the damage became more extensive. The 5-week-old flukes showed an initial stress response after 3 h, typified by blebbing of the apical plasma membrane, formation of microvilli and an accumulation and accelerated release of secretory bodies at the tegumental apex, as well as swelling of the basal infolds, The swelling increased in extent with progressively longer periods of incubation in DAMD, leading to extreme edema and sloughing of the tegument after 9 h. The 3-week-old flukes showed a stress response and swelling of the basal infolds after only 1.5 h, although sloughing of the tegument did not occur until after 9 h. In the freshly-excysted metacercaria, a stress response and some sloughing of the tegument were evident after only 0.5 h. At all stages of development, the ventral tegument was more severely affected than the dorsal, Changes also occurred to the tegumental cells which were indicative of a disruption in the synthesis and release of tegumental secretory bodies: the amount of GER became reduced, the cisternae became swollen and their ribosomal covering decreased, the Golgi complexes disappeared from the cells and the numbers of secretory bodies in the cells also decreased, The heterochromatin content of the nuclei increased and eventually the tegumental cells began to break down, Again, the changes became apparent more rapidly at the earlier stages of development. The ultrastructural changes to the tegument are linked to a possible mode of action for diamphenethide as an inhibitor of protein synthesis. In turn, the results may help to explain the drug's high efficacy against juvenile stages of F. hepatica.

FASCIOLA-HEPATICA - THE EFFECT OF THE MICROTUBULE INHIBITORS COLCHICINE AND TUBULOZOLE-C ON THE ULTRASTRUCTURE OF THE ADULT FLUKE

The effect of the microtubule inhibitors colchicine (1 x 10(-3) M) and tubulozole-C(1 x 10(-6) M) on the ultrastructure of adult Fasciola hepatica has been determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. With colchicine treatment, the apical membrane of the tegument became increasingly convoluted and blebbed, while accumulations of T1 secretory bodies occurred in the basal region of the syncytium, leading to progressively fewer secretory bodies in the syncytium. In the tegumental cells there were distinct accumulations of Tl secretory bodies around the Golgi complexes, which remained active for up to 12 h incubation. Tubulozole-treated flukes showed more severe effects, with initial accumulations of secretory bodies, both at the tegumental apex and base. This was followed in the later time-periods by the sloughing of the tegumental syncytium. In the underlying tegumental cells, the granular endoplasmic reticulum (GER) cisternae were swollen and disrupted, becoming concentrated around the nucleus. The Golgi complexes were dispersed to the periphery of the cells and gradually disappeared from the cytoplasm. After treatment with both drugs, the cell population in the vitelline follicles was altered, with an abnormally large proportion of stem cells and relatively few intermediate type 1 cells. The nurse cell cytoplasm became fragmented and was no longer in contact with the vitelline cells, while the shell globule clusters within the intermediate type 2 and mature cells were loosely packed. In the mature vitelline cells, 'yolk' globules and glycogen deposits became fewer than normal and lipid droplets were observed. The results are discussed in relation to the different modes of action of the two drugs and potential significance of this to anthelmintic (benzimidazole) therapy.

FASCIOLA-HEPATICA - THE EFFECT OF THE MICROFILAMENT INHIBITOR CYTOCHALASIN-B ON THE ULTRASTRUCTURE OF THE ADULT FLUKE

The effect of the microfilament inhibitor cytochalasin B (10 and 100-mu-g/ml) on the ultrastructure of adult Fasciola hepatica was determined in vitro by scanning and transmission electron microscopy (SEM, TEM) using both intact flukes and tissue-slice material. SEM revealed that initial swelling of the tegument led to surface blebbing and limited areas of sloughing after 24 h treatment at 100-mu-g/ml. In the tegumental syncytium, basal accumulations of secretory bodies (especially T2s) were evident in the earlier time periods but declined with longer incubations, until few secretory bodies remained in the syncytium overall. Blebbing of the apical plasma membrane and occasional areas of breakdown and sloughing of the tegument were observed over longer periods of treatment at 100-mu-g/ml. In the tegumental cell bodies, the Golgi complexes gradually decreased in size and activity, and few secretory bodies were produced. In the later time periods, the cells assumed abnormal shapes, the cytoplasm shrinking in towards the nucleus. In the vitelline follicles, a random dispersion of shell protein globules was evident within the intermediate-type cells, rather than their being organized into distinct shell globule clusters. Disruption of this process was more severe at the higher concentration of 100-mu-g/ml and again was more evident in tissue-slice material. In the latter, after prolonged (12 h) exposure to cytochalasin B, the intermediate and mature vitelline cells were filled with loosely packed and expanded shell globule clusters, containing few shell protein globules. The mature vitelline cells continued to lay down "yolk" globules and glycogen deposits. Disruption of the network of processes from the nurse cells was evident at the higher concentration of cytochalasin. Spaces began to appear between the vitelline cells and grew larger with progressively longer incubation periods, and the cells themselves assumed abnormal shapes. A number of binucleate stem cells were observed in tissue-slice material at the longest incubation period (12 h).