Purinergic system in cancer stem cells.

Accumulating evidence supports the idea that cancer stem cells (CSCs) are those with the capacity to initiate tumors, generate phenotypical diversity, sustain growth, confer drug resistance, and orchestrate the spread of tumor cells. It is still controversial whether CSCs originate from normal stem cells residing in the tissue or cancer cells from the tumor bulk that have dedifferentiated to acquire stem-like characteristics. Although CSCs have been pointed out as key drivers in cancer, knowledge regarding their physiology is still blurry; thus, research focusing on CSCs is essential to designing novel and more effective therapeutics. The purinergic system has emerged as an important autocrine-paracrine messenger system with a prominent role at multiple levels of the tumor microenvironment, where it regulates cellular aspects of the tumors themselves and the stromal and immune systems. Recent findings have shown that purinergic signaling also participates in regulating the CSC phenotype. Here, we discuss updated information regarding CSCs in the purinergic system and present evidence supporting the idea that elements of the purinergic system expressed by this subpopulation of the tumor represent attractive pharmacological targets for proposing innovative anti-cancer therapies.

Guía de Procedimientos Operacionales y Recomendaciones en la Implementación de Secuenciación de Nueva Generación para Diagnóstico Clínico / Standard Operational Procedures and Rec ommendations on Next Generation Sequenc ing for Clinic al Diagnostics

La tecnología diagnóstica conocida como NGS por sus siglas en inglés, o Secuenciación de nueva generación, es relativamente nueva, y se está implementando en algunos hospitales de Panamá. Esta tecnología ha demostrado ser una herramienta muy eficiente para la detección de alteraciones genómicas o exómicas, tanto para la clínica como para la investigación. Dada la complejidad de esta prueba, requiere una infraestructura y un número importante de recursos humanos capacitados para poder implementar esta prueba. El propósito de este documento es establecer un marco de referencia para los procedimientos administrativos en cuanto a la realización de las pruebas de secuenciación por metodología NGS. Además, que el mismo sirva de guía para el establecimiento adecuado de programas internos de control y evaluación de calidad de esta tecnología en nuestro país y la región

The technology known as Next­Generation sequencing is relatively new, and it is been implemented in some Panamanian hospitals. It has demonstrated to be a very efficient tool to identify genomic and exomic variants in a clinical setting, as well for research purposes. Because of its complexity, it requires an important infrastructure and a significant number of trained health­care professionals to carry out this test. The purpose of this document is to establish a frame for the administrative and tec hnical aspects for NGS in a clinical setting. Moreover, it will serve as a guide to establish quality control procedures that the technology requires in our country and the region.

Acute oral Bryostatin-1 administration improves learning deficits in the APP/PS1 transgenic mouse model of Alzheimer's disease.

BACKGROUND: Previous studies showed that Bryostatin-1, a potent PKC modulator and alphasecretase activator, can improve cognition in models of Alzheimer's disease (AD) with chronic (>10 weeks), intraperitoneal (i.p.) administration of the drug. We compared learning and spatial memory in the APPswe, PSEN1dE985Dbo (APP/PS1) mouse model of AD and studied the ability of acute intraperitoneal and oral Bryostatin-1 to reverse cognitive deficits in this model. Compared to wild-type (WT) mice, APP/PS1 mice showed significant delays in learning the location of a submerged platform in the Morris water maze. Bryostatin-1 was administered over a 2-week course prior to and during water maze testing. RESULTS: Acute i.p. Bryostatin-1 administration did not improve latency to escape but oral Bryostatin-1 significantly improved memory (measured by a reduction in latency to escape). This benefit of oral Bryostatin-1 administration was most apparent during the first 3 days of testing. These findings show that: 1) Bryostatin-1 is orally active in models of learning and memory, 2) this effect can be produced in less than 2 weeks and 3) this effect is not seen with i.p. administration. We conclude that oral Bryostatin-1 represents a novel, potent and long-acting memory enhancer with future clinical applications in the treatment of human AD.

Differential effects of the EGF family of growth factors on protein secretion, MAPK activation, and intracellular calcium concentration in rat lacrimal gland.

The purpose of this study was to investigate the expression of the EGF family of growth factors and EGF receptor subtypes (ErbB1-4) present in lacrimal gland and determine the effects of these growth factors on different functions of rat lacrimal gland. RT-PCR was used to detect mRNA expression in the lacrimal gland of selected members of the EGF family of growth factors, namely EGF, transforming growth factor alpha (TGF-alpha), heparin-binding EGF (HB-EGF), and heregulin. The presence of ErbB receptors was investigated by immunofluorescence microscopy and western blot analysis. The effects of EGF, TGF-alpha, HB-EGF, and heregulin on protein secretion from lacrimal gland acini were examined using a fluorescent assay for peroxidase, a marker of protein secretion. Fura-2 tetra-acetoxymethyl ester was used to measure the effects of the growth factors on intracellular [Ca2+] ([Ca2+]i) in acini. MAPK activation in acini by these growth factors was also examined by western blot analysis using antibodies specific to phosphorylated p42/44 MAPK and total p42 MAPK. Rat lacrimal gland expressed EGF, TGF-alpha, HB-EGF, and heregulin mRNA, and all four ErbB receptors were present in the lacrimal gland as detected by western blot analyses. ErbB 1 and ErbB2 were located in basal and lateral membranes of acinar and ductal cells. The location of ErbB3 could not be determined while ErbB4 was found in ductal cells. Heregulin (10(-7) m) significantly increased protein secretion in lacrimal gland acini whereas all growth factors tested significantly increased [Ca2+]i at 10(-7) m. TGF-alpha (10(-9) m), heregulin (10(-7) m), EGF (10(-7) m), and HB-EGF (10(-7) m) significantly increased the amount of phosphorylated MAPK in lacrimal gland acini. We conclude that all members of the EGF family of growth factors studied are synthesised in rat lacrimal gland, could activate all four ErbB receptors that are present in this tissue, and differentially activate lacrimal gland functions.

Presence of nerves and their receptors in mouse and human conjunctival goblet cells.

PURPOSE: To determine whether neural pathways for controlling goblet cell secretion are present in mouse and human conjunctiva. METHODS: Mouse conjunctiva was homogenized and subjected to electrophoresis and Western blotting to detect PGP 9.5 (indicates nerves), muscarinic receptor subtypes (indicates parasympathetic pathway), and adrenergic receptors (indicates sympathetic pathway). Mouse eyes and human conjunctival tissue were analyzed by immunofluorescence microscopy. Antibodies to vasoactive intestinal peptide (VIP), tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and muscarinic and alpha(1)- and beta-adrenergic receptor subtypes were used. RESULTS: Western blot demonstrated PGP 9.5, M(1), M(2), and M(3) muscarinic receptors and alpha(1A)-, beta(1)-, beta(2)-, and beta(3)-adrenergic receptors in mouse conjunctiva. Immunoreactivity for VIP, TH, and DBH was found adjacent to mouse and human goblet cells. M(1) and M(2) muscarinic receptors were identified throughout mouse conjunctiva, but M(3) receptor was predominantly on goblet cells. All three muscarinic receptor subtypes were detected on goblet cells in human conjunctiva. alpha(1A)-Adrenergic receptors were found on epithelial cells and on goblet cells in mouse and human conjunctiva. beta(1)- and beta(2)-Adrenergic receptors were found on both epithelial and goblet cells in mouse conjunctiva, but not on human conjunctival cells. beta(3)-Adrenergic receptors were found on both epithelial and goblet cells in human conjunctiva but not on mouse conjunctival cells. CONCLUSIONS: The following conclusions were drawn: parasympathetic nerves and M(1), M(2), and M(3) muscarinic receptors, as well as sympathetic nerves are present on mouse and human goblet cells. The adrenergic receptors beta(1) and beta(2,) but not alpha(1A) and beta(3) are present on mouse conjunctival goblet cells, whereas alpha(1A) and beta(3,) but not beta(1) and beta(2) are present on human conjunctival goblet cells, suggesting that these nerves and receptors could activate goblet cell secretion in mouse and humans.

Isolation, characterization, and propagation of rat conjunctival goblet cells in vitro.

PURPOSE: To isolate, culture, and characterize goblet cells from the conjunctiva of rats. METHODS: Conjunctival tissue was surgically removed from Sprague-Dawley rats. Goblet cells were then isolated from the nictitating membrane and fornix using explant cultures. Cells derived from the explants were grown and propagated in RPMI medium supplemented with 10% fetal bovine serum. They were characterized using an enzyme-linked lectin assay (ELLA) with the lectin Ulex europaeus agglutinin-1 (UEA-1), Western blot analysis, PCR, light and electron microscopy, specialized histochemistry and indirect immunofluorescence microscopy. RESULTS: Goblet cells were successfully isolated from conjunctival explants by scraping nongoblet cells from the culture vessel. To date, cultures have been passaged a minimum of three times without the loss of their specific cellular markers. Cells identified as goblet cells fulfilled the following criteria: positive staining for alcian blue/periodic acid Schiff reagent, cytokeratin (CK)-7, the lectins UEA-I and Helix pomatia agglutinin (HPA), MUC5AC, and M(3) muscarinic receptor; detection of MUC5AC mRNA using RT-PCR; and negative staining for CK-4, M(1) muscarinic receptor, and Banderia simplicifolia lectin. The authors also measured, using the ELLA, substantial amounts of UEA-I-detectable high-molecular-weight glycoproteins and MUC5AC released into the medium. CONCLUSIONS: Cultured goblet cells retain many characteristics of goblet cells in vivo and thus may serve as a useful tool in delineating the pathobiology of the ocular surface.

Regulation of conjunctival goblet cell secretion by Ca(2+)and protein kinase C.

Conjunctival goblet cells secrete mucus in response to cholinergic (muscarinic) agonists, but the underlying signaling pathways activated in this tissue are not well understood. Cholinergic agonists usually activate phospholipase C to produce inositol 1,4,5 trisphosphate and diacylglycerol. Inositol 1,4,5 trisphosphate increases the intracellular Ca(2+)concentration ([Ca2(+)](i)) while diacylglycerol activates protein kinase C (PKC). PKC and Ca(2+), either by itself or with calmodulin, activate cellular functions. Goblet cell glycoprotein secretion, our index of mucin secretion, was measured from pieces of rat conjunctiva with an enzyme-linked lectin assay using the lectin Ulex europaeus agglutinin I (UEA-I). UEA-I selectively recognizes high molecular weight glycoproteins secreted by the goblet cells. Increasing the [Ca(+)](i)with the Ca(2+)ionophore ionomycin stimulated glycoprotein secretion from conjunctival goblet cells. Cholinergic agonist-induced secretion was completely blocked by chelation of extracellular Ca(2+)and by the Ca(2+)/calmodulin-dependent protein kinase inhibitors KN93 and W7 as well as their inactive analogs KN92 and W5. Activation of classical and novel PKC isozymes by phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate stimulated goblet cell glycoprotein secretion. When ionomycin and PMA were added simultaneously, secretion was additive. PKC isozymes were identified by Western blotting analyses with antibodies specific to nine of the 11 PKC isozymes (PKCgamma and zeta were not tested). All nine PKC isozymes were identified in the conjunctival epithelium. The cellular location of the PKC isozymes was determined by immunofluorescence microscopy. Goblet cells contained the classical PKC isozymes PKCalpha, -betaI and -betaII, the novel PKC isozymes PKCepsilon, -theta;, and - mu, and the atypical PKC isozyme PKCzeta. We were unable to determine if PKC activation is required for cholinergic-agonist induced secretion because the PKC inhibitors chelerythrine and staurosporine alone greatly increased secretion. We conclude that Ca(2+)plays a major role in cholinergic agonist-induced conjunctival goblet cell secretion, but this agonist appears not to use Ca(2+)/calmodulin-dependent protein kinases. We also conclude that activated PKC can stimulate goblet cell secretion and that seven different PKC isoforms are present in the goblet cells.

Development of conjunctival goblet cells and their neuroreceptor subtype expression.

PURPOSE: To investigate expression of muscarinic, cholinergic, and adrenergic receptors on developing conjunctival goblet cells. METHODS: Eyes were removed from rats 9 to 60 days old, fixed, and used for microscopy. For glycoconjugate expression, sections were stained with Alcian blue/periodic acid-Schiffs reagent (AB/PAS) and with the lectins Ulex europeus agglutinin I (UEA-I) and Helix pomatia agglutinin (HPA). Goblet cell bodies were identified using anti-cytokeratin 7 (CK7). Nerve fibers were localized using anti-protein gene product 9.5. Location of muscarinic and adrenergic receptors was investigated using anti-muscarinic and beta-adrenergic receptors. RESULTS: At days 9 and 13, single apical cells in conjunctival epithelium stained with AB/PAS, UEA-I, and CK7. At days 17 and 60, increasing numbers of goblet cells were identified by AB/PAS, UEA-I, HPA, and CK7. Nerve fibers were localized around stratified squamous cells and at the epithelial base at days 9 and 13, and around goblet cells and at the epithelial base at days 17 and 60. At days 9 and 13, M2- and M3-muscarinic and beta2-adrenergic receptors were found in stratified squamous cells, but M1-muscarinic and beta1-adrenergic receptors were not detected. At days 17 and 60, M2- and M3-muscarinic receptors were found in goblet cells, whereas M1-muscarinic receptors were in stratified squamous cells. Beta1- and beta2-adrenergic receptors were found on both cell types. Beta3-adrenergic receptors were not detected. CONCLUSIONS: In conjunctiva, nerves, M2- and M3-muscarinic, and beta1- and beta2-adrenergic receptors are present on developing goblet cells and could regulate secretion as eyelids open.

Immunolocalization of muscarinic and VIP receptor subtypes and their role in stimulating goblet cell secretion.

PURPOSE: To determine the subtypes of cholinergic muscarinic receptors and receptors for vasoactive intestinal peptide (VIP) present in rat conjunctival goblet cells and whether cholinergic agonists and VIP stimulate goblet cell secretion. METHODS: Immunofluorescence studies were performed using antibodies against the m1, m2, and m3 muscarinic receptor subtypes and VIP receptors 1 and 2 (VIPR1 and VIPR2). The lectin Ulex europeus agglutinin I was used to measure glycoconjugate secretion, the index of secretion, from goblet cells in an enzyme-linked lectin assay. In this assay, pieces of conjunctiva were placed on filter paper and incubated for 15 to 120 minutes, with or without increasing concentrations of the cholinergic agonist carbachol or VIP. The muscarinic antagonist atropine and the muscarinic receptor-subtype-selective antagonists pirenzepine (M1), gallamine (M2), and 4-4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP mustard; M3) were incubated with carbachol to determine specificity of receptor activation. RESULTS: Immunoreactivity to M2 and M3 receptors was found on goblet cell membranes subjacent to the secretory granules. Immunoreactivity to M1 receptor was not on goblet cells but was on the stratitfied squamous cells. Immunoreactivity to VIPR2 was found on goblet cells with a localization similar to that of the M2 and M3 receptors. VIPR1 was not found on goblet cells or on the stratified squamous cells. Carbachol and VIP induced a time- and concentration-dependent stimulation of glycoconjugate secretion. Carbachol, at 10(-4) M, induced a threefold increase in glycoconjugate secretion, which was completely inhibited by atropine (10(-5) M). Carbachol-induced secretion was inhibited 54% +/- 8% by pirenzepine (10(-5) M), 69% +/- 14% by gallamine (10(-5) M), and 72% +/- 11% by 4-DAMP mustard (10(-5) M). A twofold increase in glycoconjugate secretion was obtained with VIP at 10(-8) M. CONCLUSIONS: Cholinergic agonists, through M2 and/or M3 muscarinic receptors, and VIP, through VIPR2, regulate conjunctival goblet cell secretion, suggesting that goblet cell secretion in vivo is under the control of parasympathetic nerves.