Effect of human telomerase reverse transcriptase transfection on differentiation in BeWo choriocarcinoma cells

Arrest of proliferation is one of the prerequisites for differentiation of cytotrophoblasts into syncytiotrophoblasts, and thus during differentiation telomerase activity, as well as human telomerase reverse transcriptase (hTERT) expression, is down-regulated. Considering this, it is of interest to investigate whether syncytium formation can be delayed by prolonging the expression of telomerase in cytotrophoblasts. BeWo cells were transfected with pLPC-hTERT retroviral vector and the reverse transcription-polymerase chain reaction analysis for hTERT mRNA concentrations in the transfected cells revealed a several-fold increase in hTERT mRNA compared with the cells transfected with empty vector, and this confirmed that the transfection was successful. An increase in the proliferation, as assessed by bromodeoxyuridine incorporation assay, as well as an increase in mRNA and protein concentration of various cyclins and proliferating cell nuclear antigen, was noticed. The effect of hTERT transfection was also assessed after the addition of forskolin to induce differentiation and it was observed that cell–cell fusion was delayed and differentiation did not occur in hTERT-transfected cells. However, the effects seen were only transient as stable transfection was not possible and the cells were undergoing apoptosis after 72 h, which suggested that apart from hTERT other factors might be important for immortalization of BeWo cells.

Expression Profiling of Nucleotide Metabolism-Related Genes in Human Breast Cancer Cells After Treatment with 5-Fluorouracil

5-Fluorouracil (5-FU) is one of the most widely used drugs for treatment of cancers, including breast cancer that exhibits its anticancer activity by inhibiting DNA synthesis and also incorporated into DNA and RNA. The objective of this investigation was to find out the total nucleotide metabolism genes regulated by 5-FU in breast cancer cell line. The breast cancer cell line MCF-7 was treated with the drug 5-FU. To analyze the expression of genes, we have conducted the experiment using 1.7k and 19k human microarray slide and confirmed the expression of genes by semiquantitative reverse transcription-polymerase chain reaction. The expression of 44 genes involved in the nucleotide metabolism pathway was quantified. Of these 44 genes analyzed, transcription of 6 genes were upregulated and 9 genes were downregulated. Earlier studies revealed that the transcription of genes for key enzymes like thymidylate synthase, thymidinekinase, and dihydropyrimidine dehydrogenase are regulated by 5-FU. This study identified some novel genes like thioredoxin reductase, ectonucleotide triphosphate dephosphorylase, and CTP synthase are regulated by 5-FU. The data also reveal large-scale perturbation in transcription of genes not involved directly in the known mechanism of action of 5-FU.

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.

Regulation of extracellular matrix genes by arecoline in primary gingival fibroblasts requires epithelial factors

Background and Objective: Oral submucous fibrosis, a disease of collagen disorder, has been attributed to arecoline present in the saliva of betel quid chewers. However, the molecular basis of the action of arecoline in the pathogenesis of oral submucous fibrosis is poorly understood. The basic aim of our study was to elucidate the mechanism underlying the action of arecoline on the expression of genes in oral fibroblasts. Material and Methods: Human keratinocytes (HaCaT cells) and primary human gingival fibroblasts were treated with arecoline in combination with various pathway inhibitors, and the expression of transforming growth factor-beta isoform genes and of collagen isoforms was assessed using reverse transcription polymerase chain reaction analysis. Results: We observed the induction of transforming growth factor-beta2 by arecoline in HaCaT cells and this induction was found to be caused by activation of the M-3 muscarinic acid receptor via the induction of calcium and the protein kinase C pathway. Most importantly, we showed that transforming growth factor-beta2 was significantly overexpressed in oral submucous fibrosis tissues (p = 0.008), with a median of 2.13 (n = 21) compared with 0.75 (n = 18) in normal buccal mucosal tissues. Furthermore, arecoline down-regulated the expression of collagens 1A1 and 3A1 in human primary gingival fibroblasts; however these collagens were induced by arecoline in the presence of spent medium of cultured human keratinocytes. Treatment with a transforming growth factor-beta blocker, transforming growth factor-beta1 latency-associated peptide, reversed this up-regulation of collagen, suggesting a role for profibrotic cytokines, such as transforming growth factor-beta, in the induction of collagens. Conclusion: Taken together, our data highlight the importance of arecoline-induced epithelial changes in the pathogenesis of oral submucous fibrosis.

Performance of RT-PCR-ELISA for the detection of peste des petits ruminants virus

A highly sensitive and specific reverse transcription polymerase chain reaction enzyme linked immunosorbent assay (RT-PCR-ELISA) was developed for the objective detection of nucleoprotein (N) gene of peste des petits ruminants (PPR) virus from field outbreaks or experimentally infected sheep. Two primers (IndF and Np4) and one probe (Sp3) available or designed for the amplification/probing of the 'N' gene of PPR virus, were chosen for labeling and use in RT-PCR-ELISA based on highest analytical sensitivity of detection of infective virus or N-gene containing recombinant plasmid, higher nucleotide homology at the primer binding sites of the 'N' gene sequences available and the ability to amplify PPR viral genome from different sources of samples. RT-PCR was performed with unlabeled IndF and Np4 digoxigenin labeled primers followed by a microplate hybridization probe reaction with biotin labeled Sp3 probe. RT-PCR-ELISA was found to be 10-fold more sensitive than the conventional RT-PCR followed by agarose gel based detection of PCR product. Based on the Mean (mean +/- 3S.D.) optical density (OD) values of 47 RT-PCR negative samples, OD values above 0.306 were considered positive in RT-PCR-ELISA. A total of 82 oculo-nasal swabs and tissue samples from suspected PPR cases were analyzed by RT-PCR and RT-PCR-ELISA, which revealed 54.87 and 58.54% positivity, respectively. From an experimentally infected sheep, both RT-PCR and RT-PCR-ELISA could detect the virus from 6 days post-infection up to 9 days in oculo-nasal swabs. On post-mortem, PPR viral genome was detected in spleen, lymph node, lung, heart and liver. The correlation co-efficient between RT-PCR-ELISA OD values and either TCID50 of virus or molecules of DNA was 0.622 and 0.657, respectively. The advantages of RT-PCR-ELISA over the conventional agarose gel based detection of RT-PCR products are discussed. (c) 2006 Elsevier B.V. All rights reserved.

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.

Development of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of sugarcane streak mosaic virus isolates from Southern India

Sugarcane streak mosaic virus (SCSMV), causes mosaic disease of sugarcane and is thought to belong to a new undescribed genus in the family Potyviridae. The coat protein (CP) gene from the Andhra Pradesh (AP) isolate of SCSMV (SCSMV AP) was cloned and expressed in Escherichia coli. The recombinant coat protein was used to raise high quality antiserum. The CP antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection and discrimination of SCSMV isolates in South India. The sequence of the cloned PCR products encoding 3'untranslated region (UTR) and CP regions of the virus isolates from three different locations in South India viz. Tanuku (Coastal Andhra Pradesh), Coimbatore (Tamil Nadu) and Hospet (Karnataka) was compared with that of SCSMV AP The analysis showed that they share 89.4, 89.5 and 90% identity respectively at the nucleotide level. This suggests that the isolates causing mosaic disease of sugarcane in South India are indeed strains of SCSMV In addition, the sensitivity of the IC-RT-PCR was compared with direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA) and dot-blot immunobinding assays and was found to be more sensitive and hence could be used to detect the presence of virus in sugarcane breeding, germplasm centres and in quarantine programs.

Luteinizing hormone regulates inhibin-alpha subunit expression through multiple signaling pathways involving steroidogenic factor-1 and beta-catenin in the macaque corpus luteum

We employed different experimental model systems to define the role of GATA4, beta-catenin, and steroidogenic factor (SF-1) transcriptional factors in the regulation of monkey luteal inhibin secretion. Reverse transcription polymerase chain reactions and western blotting analyses show high expression of inhibin-alpha, GATA4, and beta-catenin in corpus luteum (CL) of the mid-luteal phase. Gonadotropin-releasing hormone receptor antagonist-induced luteolysis model suggested the significance of luteinizing hormone (LH) in regulating these transcriptional factors. Inducible cyclic AMP early repressor mRNA expression was detected in the CL and no change was observed in different stages of CL. Following amino acid sequence analysis, interaction between SF-1 and beta-catenin in mid-stage CL was verified by reciprocal co-immunoprecipitation experiments coupled to immunoblot analysis. Electrophoretic mobility shift analysis support the role of SF-1 in regulating luteal inhibin-alpha expression. Our results suggest a possible multiple crosstalk of Wnt, cAMP, and SF-1 in the regulation of luteal inhibin secretion.

STAT-1 expression is regulated by IGFBP-3 in malignant glioma cells and is a strong predictor of poor survival in patients with glioblastoma Laboratory investigation

Object. Insulin-like growth factor binding proteins (IGEBPs) have been implicated in the pathogenesis of glioma. In a previous study the authors demonstrated that IGFBP-3 is a novel glioblastoma biomarker associated with poor survival. Since signal transducer and activator of transcription 1 (STAT-1) has been shown to be regulated by IGFBP-3 during chondrogenesis and is a prosurvival and radioresistant molecule in different tumors, the aim in the present study was to explore the functional significance of IGFBP-3 in malignant glioma cells, to determine if STAT-1 is indeed regulated by IGFBP-3, and to study the potential of STAT-1 as a biomarker in glioblastoma. Methods. The functional significance of IGFBP-3 was investigated using the short hairpin (sh)RNA gene knockdown approach on U251MG cells. STAT-1 regulation by IGFBP-3 was tested on U251MG and U87MG cells by shRNA gene knockdown and exogenous treatment with recombinant IGFBP-3 protein. Subsequently, the expression of STAT-1 was analyzed with real-time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) in glioblastoma and control brain tissues. Survival analyses were done on a uniformly treated prospective cohort of adults with newly diagnosed glioblastoma (136 patients) using Kaplan-Meier and Cox regression models. Results. IGFBP-3 knockdown significantly impaired proliferation, motility, migration, and invasive capacity of U251MG cells in vitro (p < 0.005). Exogenous overexpression of IGFBP-3 in U251MG and U87MG cells demonstrated STAT-1 regulation. The mean transcript levels (by real-time RT-PCR) and the mean labeling index of STAT-1 (by IHC) were significantly higher in glioblastoma than in control brain tissues (p = 0.0239 and p < 0.001, respectively). Multivariate survival analysis revealed that STAT-1 protein expression (HR 1.015, p = 0.033, 95% CI 1.001-1.029) along with patient age (HR 1.025, p = 0.005, 95% CI 1.008-1.042) were significant predictors of shorter survival in patients with glioblastoma. Conclusions. IGFBP-3 influences tumor cell proliferation, migration, and invasion and regulates STAT-1 expression in malignant glioma cells. STAT-1 is overexpressed in human glioblastoma tissues and emerges as a novel prognostic biomarker.

Molecular mechanism for the specific inhibition of reverse transcriptase of rous sarcoma virus by the copper complexes of isonicotinic acid hydrazide

Isonicotinic acid hydrazide (isoniazid), one of the most potent antitubercular drugs, was recently shown, in our laboratory, to form two different complexes with copper, depending upon the oxidation state of the metal ion. Both the complexes have been shown to possess antiviral activity against Rous sarcoma virus, an RNA tumor virus. The antiviral activity of the complexes has been attributed to their ability to inhibit the endogenous reverse transcriptase activity of RSV. More recent studies in our laboratory indicate that both these complexes inhibit both endogenous and exogenous reactions. As low a final concentration as 50 μM of the cupric and the cuprous complexes inhibits the endogenous reaction to the extent of 93 and 75 per cent respectively. Inhibition of the exogenous reaction varies with the templates. The inhibition can be reversed by either β-mercaptoethanol or ethylene-diamine-tetra-acetic acid. The specificity of this inhibition has been ascertained by using a synthetic primer-template, −(dG)not, vert, similar15−(rCm)n, which is highly specific for reverse transcriptases. The inhibition is found to be template specific. The studies carried out, using various synthetic primer-templates, show the inhibition of both the steps of reverse transcription by the copper complexes of isoniazid.

Novel flutamide regulated genes in the rat ventral prostate: differential modulation of their expression by castration and flutamide treatments

Aim: To identify flutamide regulated genes in the rat ventral prostate. Methods: Total RNA from ventral prostates control and flutamide treated rats were isolated. Differentially expressed transcripts were identified using display reverse transcriptase polymerase chain reaction. The effect of castration on the expression of regulated transcripts was studied. Results: We have identified beta 2-microglobulin, cytoplasmic FMR1 protein 2 and pumilio 1 as flutamide induced and spermine binding protein and ribophorin II as flutamide targets in the rat ventral prostate. Although flutamide treatment caused an induction of pumilio I mRNA, had no effect. Conclusion: Castration and flutamide treatments exert differential effects on gene expression. might also have direct AR independent effects, which might have implications in the emergence of androgen dent prostate cancer and the failure of flutamide therapy.

Glioblastoma-Specific Protein Interaction Network Identifies PP1A and CSK21 as Connecting Molecules between Cell Cycle-Associated Genes

Glioblastoma (GBM; grade IV astrocytoma) is a very aggressive form of brain cancer with a poor survival and few qualified predictive markers. This study integrates experimentally validated genes that showed specific upregulation in GBM along with their protein-protein interaction information. A system level analysis was used to construct GBM-specific network. Computation of topological parameters of networks showed scale-free pattern and hierarchical organization. From the large network involving 1,447 proteins, we synthesized subnetworks and annotated them with highly enriched biological processes. A careful dissection of the functional modules, important nodes, and their connections identified two novel intermediary molecules CSK21 and protein phosphatase 1 alpha (PP1A) connecting the two subnetworks CDC2-PTEN-TOP2A-CAV1-P53 and CDC2-CAV1-RB-P53-PTEN, respectively. Real-time quantitative reverse transcription-PCR analysis revealed CSK21 to be moderately upregulated and PP1A to be overexpressed by 20-fold in GBM tumor samples. Immunohistochemical staining revealed nuclear expression of PP1A only in GBM samples. Thus, CSK21 and PP1A, whose functions are intimately associated with cell cycle regulation, might play key role in gliomagenesis. Cancer Res; 70(16); 6437-47. (C)2010 AACR.

Progesterone receptor expression in the human placenta

The presence of progesterone receptors (PR) in the human placenta has been demonstrated using the reverse transcriptase-polymerase chain reaction technique. It was observed that the amount of PR in the human placenta is less during late gestation. Electrophoretic mobility shift assays with nuclear extract isolated from the first trimester and term placenta revealed three complexes when incubated with [P-32]dCTP-labelled progesterone response element, and, in competition with unlabelled progesterone response element, the formation of all three complexes was inhibited. When supershift analysis of these complexes was carried out using antibodies which cross-react with both the A and B types of the PR or only with the B type receptor, only the A-form of PR was detected in the human placenta.

Whole exome sequencing identifies a novel splice-site mutation in ADAMTS17 in an Indian family with Weill-Marchesani syndrome

Purpose: Weill-Marchesani syndrome (WMS) is a rare connective tissue disorder, characterized by short stature, micro-spherophakic lens, and stubby hands and feet (brachydactyly). WMS is caused by mutations in the FBN1, ADAMTS10, and LTBP2 genes. Mutations in the LTBP2 and ADAMTS17 genes cause a WMS-like syndrome, in which the affected individuals show major features of WMS but do not display brachydactyly and joint stiffness. The main purpose of our study was to determine the genetic cause of WMS in an Indian family. Methods: Whole exome sequencing (WES) was used to identify the genetic cause of WMS in the family. The cosegregation of the mutation was determined with Sanger sequencing. Reverse transcription (RT)-PCR analysis was used to assess the effect of a splice-site mutation on splicing of the ADAMTS17 transcript. Results: The WES analysis identified a homozygous novel splice-site mutation c.873+1G>T in a known WMS-like syndrome gene, ADAMTS17, in the family. RT-PCR analysis in the patient showed that exon 5 was skipped, which resulted in the deletion of 28 amino acids in the ADAMTS17 protein. Conclusions: The mutation in the WMS-like syndrome gene ADAMTS17 also causes WMS in an Indian family. The present study will be helpful in genetic diagnosis of this family and increases the number of mutations of this gene to six.

Intermittent electrical stimuli for guidance of human mesenchymal stem cell lineage commitment towards neural-like cells on electroconductive substrates

In the context of the role of multiple physical factors in dictating stem cell fate, the present paper demonstrates the effectiveness of the intermittently delivered external electric field stimulation towards switching the stem cell fate to specific lineage, when cultured in the absence of biochemical growth factors. In particular, our findings present the ability of human mesenchymal stem cells (hMSCs) to respond to the electric stimuli by adopting extended neural-like morphology on conducting polymeric substrates. Polyaniline (PANI) is selected as the model system to demonstrate this effect, as the electrical conductivity of the polymeric substrates can be systematically tailored over a broad range (10(-9) to 10 S/cm) from highly insulating to conducting by doping with varying concentrations (10(-5) to 1 M) of HCl. On the basis of the culture protocol involving the systematic delivery of intermittent electric field (dc) stimulation, the parametric window of substrate conductivity and electric field strength was established to promote significant morphological extensions, with minimal cellular damage. A time dependent morphological change in hMSCs with significant filopodial elongation was observed after 7 days of electrically stimulated culture. Concomitant with morphological changes, a commensurate increase in the expression of neural lineage commitment markers such as nestin and PI tubulin was recorded from hMSCs grown on highly conducting substrates, as revealed from the mRNA expression analysis using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) as well as by immune-fluorescence imaging. Therefore, the present work establishes the key role of intermittent and systematic delivery of electric stimuli as guidance cues in promoting neural-like differentiation of hMSCs, when grown on electroconductive substrates. (C) 2014 Elsevier Ltd. All rights reserved.