Expression of the receptor guanylyl cyclase C and its ligands in reproductive tissues of the rat: A potential role for a novel signaling pathway in the epididymis

Guanylyl cyclase C (GC-C) is a membrane-associated form of guanylyl cyclase and serves as the receptor for the heat-stable enterotoxin (ST) peptide and endogenous ligands guanylin, uroguanylin, and lymphoguanylin. The major site of expression of GC-C is the intestinal epithelial cell, although GC-C is also expressed in extraintestinal tissue such as the kidney, airway epithelium, perinatal liver, stomach, brain, and adrenal glands. Binding of ligands to GC-C leads to accumulation of intracellular cGMP, the activation of protein kinases G and A, and phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that regulates salt and water secretion. We examined the expression of GC-C and its ligands in various tissues of the reproductive tract of the rat. Using reverse transcriptase and the polymerase chain reaction, we demonstrated the presence of GC-C, uroguanylin, and guanylin mRNA in both male and female reproductive organs. Western blot analysis using a monoclonal antibody to GC-C revealed the presence of differentially glycosylated forms of GC-C in the caput and cauda epididymis. Exogenous addition of uroguanylin to minced epididymal tissue resulted in cGMP accumulation, suggesting an autocrine or endocrine activation of GC-C in this tissue. Immunohistochemical analyses demonstrated expression of GC-C in the tubular epithelial cells of both the caput epididymis and cauda epididymis. Our results suggest that the GC-C signaling pathway could converge on CFTR in the epididymis and perhaps control fluid and ion balance for optimal sperm maturation and storage in this tissue.

Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties

Emerging evidence suggests that cancers arise in stem/progenitor cells. Yet, the requirements for transformation of these primitive cells remains poorly understood. In this study, we have exploited the `mammosphere' system that selects for primitive mammary stem/progenitor cells to explore their potential and requirements for transformation. Introduction of Simian Virus 40 Early Region and hTERT into mammosphere-derived cells led to the generation of NBLE, an immortalized mammary epithelial cell line. The NBLEs largely comprised of bi-potent progenitors with long-term self-renewal and multi-lineage differentiation potential. Clonal and karyotype analyses revealed the existence of heterogeneous population within NBLEs with varied proliferation, differentiation and sphere-forming potential. Significantly, injection of NBLEs into immunocompromised mice resulted in the generation of invasive ductal adenocarcinomas. Further, these cells harbored a sub-population of CD44(+)/CD24(-) fraction that alone had sphere- and tumor-initiating potential and resembled the breast cancer stem cell gene signature. Interestingly, prolonged in vitro culturing led to their further enrichment. The NBLE cells also showed increased expression of stemness and epithelial to mesenchymal transition markers, deregulated self-renewal pathways, activated DNA-damage response and cancer-associated chromosomal aberrations-all of which are likely to have contributed to their tumorigenic transformation. Thus, unlike previous in vitro transformation studies that used adherent, more differentiated human mammary epithelial cells our study demonstrates that the mammosphere-derived, less-differentiated cells undergo tumorigenic conversion with only two genetic elements, without requiring oncogenic Ras. Moreover, the striking phenotypic and molecular resemblance of the NBLE-generated tumors with naturally arising breast adenocarcinomas supports the notion of a primitive breast cell as the origin for this subtype of breast cancer. Finally, the NBLEs represent a heterogeneous population of cells with striking plasticity, capable of differentiation, self-renewal and tumorigenicity, thus offering a unique model system to study the molecular mechanisms involved with these processes. Oncogene (2012) 31, 1896-1909; doi:10.1038/onc.2011.378; published online 29 August 2011

Advancing Edge Speeds of Epithelial Monolayers Depend on Their Initial Confining Geometry

Collective cell migrations are essential in several physiological processes and are driven by both chemical and mechanical cues. The roles of substrate stiffness and confinement on collective migrations have been investigated in recent years, however few studies have addressed how geometric shapes influence collective cell migrations. Here, we address the hypothesis that the relative position of a cell within the confinement influences its motility. Monolayers of two types of epithelial cells-MCF7, a breast epithelial cancer cell line, and MDCK, a control epithelial cell line-were confined within circular, square, and cross-shaped stencils and their migration velocities were quantified upon release of the constraint using particle image velocimetry. The choice of stencil geometry allowed us to investigate individual cell motility within convex, straight and concave boundaries. Cells located in sharp, convex boundaries migrated at slower rates than those in concave or straight edges in both cell types. The overall cluster migration occurred in three phases: an initial linear increase with time, followed by a plateau region and a subsequent decrease in cluster speeds. An acto-myosin contractile ring, present in the MDCK but absent in MCF7 monolayer, was a prominent feature in the emergence of leader cells from the MDCK clusters which occurred every similar to 125 mu m from the vertex of the cross. Further, coordinated cell movements displayed vorticity patterns in MDCK which were absent in MCF7 clusters. We also used cytoskeletal inhibitors to show the importance of acto-myosin bounding cables in collective migrations through translation of local movements to create long range coordinated movements and the creation of leader cells within ensembles. To our knowledge, this is the first demonstration of how bounding shapes influence long-term migratory behaviours of epithelial cell monolayers. These results are important for tissue engineering and may also enhance our understanding of cell movements during developmental patterning and cancer metastasis.

Regulation of oxidative-stress responsive genes by arecoline in human keratinocytes

Background and Objective: Arecoline, an arecanut alkaloid present in the saliva of betel quid chewers, has been implicated in the pathogenesis of a variety of inflammatory oral diseases, including oral submucous fibrosis and periodontitis. To understand the molecular b asis of arecoline action in epithelial changes associated with these diseases, we investigated the effects of arecoline on human keratinocytes with respect to cell growth regulation and the expression of stress-responsive genes.Material and Methods:Human keratinocyte cells (of the HaCaT cell line) were treated with arecoline, following which cell viability was assessed using the Trypan Blue dye-exclusion assay, cell growth and proliferation were analyzed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and 5-bromo-2-deoxyuridine incorporation assays, cell cycle arrest and generation of reactive oxygen species were examined using flow cytometry, and gene expression changes were investigated using the reverse transcription-polymerase chain reaction technique. The role of oxidative stress, muscarinic acetylcholine receptor and mitogen-activated protein kinase (MAPK) pathways were studied using specific inhibitors. Western blot analysis was performed to study p38 MAPK activation.Results:Arecoline induced the generation of reactive oxygen species and cell cycle arrest at the G1/G0 phase in HaCaT cells without affecting the expression of p21/Cip1. Arecoline-induced epithelial cell death at higher concentrations was caused by oxidative trauma without eliciting apoptosis. Sublethal concentrations of arecoline upregulated the expression of the following stress-responsive genes: heme oxygenase-1; ferritin light chain; glucose-6-phosphate dehydrogenase; glutamate-cysteine ligase catalytic subunit; and glutathione reductase.Additionally, there was a dose-dependent induction of interleukin-1alfa mRNA by arecoline via oxidative stress and p38 MAPK activation. Conclusion:our data highlight the role of oxidative stress in arecoline-mediated cell death, gene regulation and inflammatory processes in human keratinocytes.

Cyclic nucleotide signaling in intestinal epithelia: getting to the gut of the matter

The intestine is the primary site of nutrient absorption, fluid-ion secretion, and home to trillions of symbiotic microbiota. The high turnover of the intestinal epithelia also renders it susceptible to neoplastic growth. These diverse processes are carefully regulated by an intricate signaling network. Among the myriad molecules involved in intestinal epithelial cell homeostasis are the second messengers, cyclic AMP (cAMP) and cyclic GMP (cGMP). These cyclic nucleotides are synthesized by nucleotidyl cyclases whose activities are regulated by extrinsic and intrinsic cues. Downstream effectors of cAMP and cGMP include protein kinases, cyclic nucleotide gated ion channels, and transcription factors, which modulate key processes such as ion-balance, immune response, and cell proliferation. The web of interaction involving the major signaling pathways of cAMP and cGMP in the intestinal epithelial cell, and possible cross-talk among the pathways, are highlighted in this review. Deregulation of these pathways occurs during infection by pathogens, intestinal inflammation, and cancer. Thus, an appreciation of the importance of cyclic nucleotide signaling in the intestine furthers our understanding of bowel disease, thereby aiding in the development of therapeutic approaches.

Potential histopathological and molecular changes in rat vas deferens inhaled by Boswellia papyrifera and Boswellia carterii

Boswellia papyrifera and Boswellia carterii released from smoke contaminate indoor environment and consequently adversely affect humans as evidenced by respiratory disturbances. The aim of this study was to determine the effects of these plants on pathological and biochemical changes in vas deferens of albino rats. Animals were administered 4g/kg body weight B. papyrifera and B. carterii daily for 120days along with controls. Significant changes were observed in epithelial cell types and some cells showed signs of degeneration. The ultrastructural studies revealed marked changes in cytoplasmic organelles. Microvilli were missing and lysosomes were found in the cytoplasm. In addition, all treated groups plasma fructose and other biochemical parameters were decreased indicating reduced energy necessary for motility and contractility of spermatozoa. Many spermatozoa were disorganized and agglomerated. Data suggest that smoke from these plants adversely affects vas deferens.

Growth inhibition of human promonocytic leukaemic U937 cells by interferon gamma is irreversible and not cell cycle phase-specific.

Growth of human promonocytic leukaemic U937 cells was found arrested within 24 h upon exposure to interferon gamma (IFN-gamma). Removal of the interferon did not result in the resumption of growth, as is evident from the absence of doubling of viable cell count and(3)H-thymidine incorporation. 5-Bromo-2'-deoxyuridine-based flow cytometric analysis of the growth-arrested cells, 24 h subsequent to the removal of IFN-gamma, showed absence of DNA synthesis, confirming the irreversible nature of the growth inhibition. Propidium iodide-based flow cytometric analysis of the growth-arrested cells showed a distribution which is typical of a growth inhibition without resulting in the accumulation of cells in any specific phase of the cell cycle. These results indicated that IFN-gamma arrested growth of U937 cells in an irreversible and cell cycle phase-independent manner. These observations were in contrast to our earlier report on the reversible and cell cycle phase-specific growth inhibition of human amniotic (fetal epithelial) WISH cells by the interferon. Copyright 1999 Academic Press.

Gene expression profile of epithelial cells and mesenchymal cells derived from limbal explant culture

Purpose: Limbal stem cell deficiency is a challenging clinical problem and the current treatment involves replenishing the depleted limbal stem cell (LSC) pool by either limbal tissue transplantation or use of cultivated limbal epithelial cells (LEC). Our experience of cultivating the LEC on denuded human amniotic membrane using a feeder cell free method, led to identification of mesenchymal cells of limbus (MC-L), which showed phenotypic resemblance to bone marrow derived mesenchymal stem cells (MSC-BM). To understand the transcriptional profile of these cells, microarray experiments were carried out.Methods: RNA was isolated from cultured LEC, MC-L and MSC-BM and microarray experiments were carried out by using Agilent chip (4x44 k). The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction. Results: The microarray analysis revealed specific gene signature of LEC and MC-L, and also their complementary role related to cytokine and growth factor profile, thus supporting the nurturing roles of the MC-L. We have also observed similar and differential gene expression between MC-L and MSC-BM.Conclusions: This study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells.

Transcription factors that mediate epithelial-mesenchymal transition lead to multidrug resistance by upregulating ABC transporters

Development of multidrug resistance (MDR) is a major deterrent in the effective treatment of metastatic cancers by chemotherapy. Even though MDR and cancer invasiveness have been correlated, the molecular basis of this link remains obscure. We show here that treatment with chemotherapeutic drugs increases the expression of several ATP binding cassette transporters (ABC transporters) associated with MDR, as well as epithelial-mesenchymal transition (EMT) markers, selectively in invasive breast cancer cells, but not in immortalized or non-invasive cells. Interestingly, the mere induction of an EMT in immortalized and non-invasive cell lines increased their expression of ABC transporters, migration, invasion, and drug resistance. Conversely, reversal of EMT in invasive cells by downregulating EMT-inducing transcription factors reduced their expression of ABC transporters, invasion, and rendered them more chemosensitive. Mechanistically, we demonstrate that the promoters of ABC transporters carry several binding sites for EMT-inducing transcription factors, and overexpression of Twist, Snail, and FOXC2 increases the promoter activity of ABC transporters. Furthermore, chromatin immunoprecipitation studies revealed that Twist binds directly to the E-box elements of ABC transporters. Thus, our study identifies EMT inducers as novel regulators of ABC transporters, thereby providing molecular insights into the long-standing association between invasiveness and MDR. Targeting EMT transcription factors could hence serve as novel strategies to curb both metastasis and the associated drug resistance. Cell Death and Disease (2011) 2, e179; doi:10.1038/cddis.2011.61; published online 7 July 2011

Potential ultrastructural changes in rat epididymal cell types induced by Boswellia papyrifera and Boswellia carterii incense

Boswellia papyrifera and Boswellia carterii, known as Arabian incense, diffuses smoke, contaminating the air, which adversely affects human health. Therefore, this study was designed to ascertain the effect of these plants on histopathological and ultrastructure changes in cauda epididymis of Albino rats. Animals were exposed to 4 g/kg body weight of B. papyrifera and B. carterii daily for 120 days along with suitable controls. Our study indicates a significant reduction in epithelial heights. Cells showed signs of degeneration. The ultrastructural study revealed that the cauda epididymis was affected, including its cell types. Furthermore, a decrease in the size of mitochondria, Golgi complex, and both ERs was observed. In all treated groups, plasma fructose decreased considerably, indicating the sign of reduced energy, vital for motility and other sperm functions. The results of this study suggest that these plants systematically affect cauda epididymal cell types and its lumen through its potential toxicity. (C) 2013 Published by Elsevier Masson SAS on behalf of Academie des sciences.

Intestinal Cell Proliferation and Senescence Are Regulated by Receptor Guanylyl Cyclase C and p21

Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c(-/-), mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence.

Differentially Selective Chemosensor with Fluorescence Off-On Responses on Cu2+ and Zn2+ Ions in Aqueous Media and Applications in Pyrophosphate Sensing, Live Cell Imaging, and Cytotoxicity

A new benzoyl hydrazone based chemosensor R is synthesized by Schiff base condensation of 2,6-diformyl-4-methylphenol and phenyl carbohydrazide and acts as a highly selective fluorescence sensor for Cu2+ and Zn2+ ions in aqueous media. The reaction of R with CuCl2 or ZnCl2 forms the corresponding dimeric dicopper(II) Cu-2(R)(CH3O)-(NO3)](2)(CH3O)(2) (R-Cu2+) and dizinc(1) Zn-2(R)(2)](NO3)(2) (R-Zn2+) complexes, which are characterized, as R, by conventional techniques including single-crystal X-ray analysis. Electronic absorption and fluorescence titration studies of R with different metal cations in a CH3CN/0.02 M HEPES buffer medium (pH = 7.3) show a highly selective binding affinity only toward Cu(2+)and Zn2+ ions even in the presence of other commonly coexisting ions such as Ne+, K+, Mg2+, Ca2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cd2+, and Hg2+. Quantification of the fluorescence titration analysis shows that the chemosensor R can indicate the presence of Cu2+ and Zn2+ even at very low concentrations of 17.3 and 16.5 ppb, respectively. R-Zn2+ acts as a selective metal-based fluorescent sensor for inorganic pyrophosphate ion (PPi) even in the presence of other common anions such as F-, Cl-, Br-, I-, CH3COO-, CO32-, HCO3-, N-3(-), SO42-, PPi, AMP, ADP, and ATP in an aqueous medium. The propensity of R as a bioimaging fluorescent probe to detect Cu2+ and Zn2+ ions in human cervical HeLa cancer cell lines and their cytotoxicity against human cervical (HeLa), breast cancer (MCF7), and noncancer breast epithelial (MCF10a) cells have also been investigated. R-Cu2+ shows better cytotoxicity and sensitivity toward cancer cells over noncancer cells than R and R-Zn2+ under identical conditions, with the appearance of apoptotic bodies.

Mycobacterium bovis BCG promotes tumor cell survival from tumor necrosis factor-alpha-induced apoptosis

Background: Increased incidence of lung cancer among pulmonary tuberculosis patients suggests mycobacteria-induced tumorigenic response in the host. The alveolar epithelial cells, candidate cells that form lung adenocarcinoma, constitute a niche for mycobacterial replication and infection. We thus explored the possible mechanism of M. bovis Bacillus Calmette-Guerin (BCG)-assisted tumorigenicity in type II epithelial cells, human lung adenocarcinoma A549 and other cancer cells. Methods: Cancer cell lines originating from lung, colon, bladder, liver, breast, skin and cervix were treated with tumor necrosis factor (TNF)-alpha in presence or absence of BCG infection. p53, COP1 and sonic hedgehog (SHH) signaling markers were determined by immunoblotting and luciferase assays, and quantitative real time PCR was done for p53-responsive pro-apoptotic genes and SHH signaling markers. MTT assays and Annexin V staining were utilized to study apoptosis. Gain-and loss-of-function approaches were used to investigate the role for SHH and COP1 signaling during apoptosis. A549 xenografted mice were used to validate the contribution of BCG during TNF-alpha treatment. Results: Here, we show that BCG inhibits TNF-alpha-mediated apoptosis in A549 cells via downregulation of p53 expression. Substantiating this observation, BCG rescued A549 xenografts from TNF-alpha-mediated tumor clearance in nude mice. Furthermore, activation of SHH signaling by BCG induced the expression of an E3 ubiquitin ligase, COP1. SHH-driven COP1 targeted p53, thereby facilitating downregulation of p53-responsive pro-apoptotic genes and inhibition of apoptosis. Similar effects of BCG could be shown for HCT116, T24, MNT-1, HepG2 and HELA cells but not for HCT116 p53(-/-) and MDA-MB-231 cells. Conclusion: Our results not only highlight possible explanations for the coexistence of pulmonary tuberculosis and lung cancer but also address probable reasons for failure of BCG immunotherapy of cancers.

Bmi1 regulates self-renewal and epithelial to mesenchymal transition in breast cancer cells through Nanog

Background: The Bmi1 polycomb ring finger oncogene, a transcriptional repressor belonging to the Polycomb group of proteins plays an important role in the regulation of stem cell self-renewal and is elevated in several cancers. In the current study, we have explored the role of Bmi1 in regulating the stemness and drug resistance of breast cancer cells. Methods: Using real time PCR and immunohistochemistry primary breast tissues were analyzed. Retro-and lentiviruses were utilized to overexpress and knockdown Bmi1, RT-PCR and Western blot was performed to evaluate mRNA and protein expression. Stemness properties were analyzed by flow cytometry and sphere-formation and tumor formation was determined by mouse xenograft experiments. Dual luciferase assay was employed to assess promoter activity and MTT assay was used to analyze drug response. Results: We found Bmi1 overexpression in 64% of grade III invasive ductal breast adenocarcinomas compared to normal breast tissues. Bmi1 overexpression in immortalized and transformed breast epithelial cells increased their sphere-forming efficiency, induced epithelial to mesenchymal transition ( EMT) with an increase in the expression of stemness-related genes. Knockdown of Bmi1 in tumorigenic breast cells induced epithelial morphology, reduced expression of stemness-related genes, decreased the IC50 values of doxorubicin and abrogated tumor-formation. Bmi1-high tumors showed elevated Nanog expression whereas the tumors with lower Bmi1 showed reduced Nanog levels. Overexpression of Bmi1 increased Nanog levels whereas knockdown of Bmi1 reduced its expression. Dual luciferase promoter-reporter assay revealed Bmi1 positively regulated the Nanog and NF kappa B promoter activity. RT-PCR analysis showed that Bmi1 overexpression activated the NF kappa B pathway whereas Bmi1 knockdown reduced the expression of NF kappa B target genes, suggesting that Bmi1 might regulate Nanog expression through the NF kappa B pathway. Conclusions: Our study showed that Bmi1 is overexpressed in several high-grade, invasive ductal breast adenocarcinomas, thus supporting its role as a prognostic marker. While Bmi1 overexpression increased self-renewal and promoted EMT, its knockdown reversed EMT, reduced stemness, and rendered cells drug sensitive, thus highlighting a crucial role for Bmi1 in regulating the stemness and drug response of breast cancer cells. Bmi1 may control self-renewal through the regulation of Nanog expression via the NF kappa B pathway.