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PURPOSE: The transcription factors ZEB1 and ZEB2 mediate epithelial-to-mesenchymal transition (EMT) and metastatic progression in numerous malignancies including breast cancer. ZEB1 and ZEB2 drive EMT through transcriptional repression of cell-cell junction proteins and members of the tumor suppressive miR200 family. However, in estrogen receptor positive (ER +) breast cancer, the role of ZEB2 as an independent driver of metastasis has not been fully investigated. METHODS: In the current study, we induced exogenous expression of ZEB2 in ER + MCF-7 and ZR-75-1 breast cancer cell lines and examined EMT gene expression and metastasis using dose-response qRT-PCR, transwell migration assays, proliferation assays with immunofluorescence of Ki-67 staining. We used RNA sequencing to identify pathways and genes affected by ZEB2 overexpression. Finally, we treated ZEB2-overexpressing cells with 17ß-estradiol (E2) or ICI 182,780 to evaluate how ZEB2 affects estrogen response. RESULTS: Contrary to expectation, we found that ZEB2 did not increase canonical epithelial nor decrease mesenchymal gene expressions. Furthermore, ZEB2 overexpression did not promote a mesenchymal cell morphology. However, ZEB1 and ZEB2 protein expression induced significant migration of MCF-7 and ZR-75-1 breast cancer cells in vitro and MCF-7 xenograft metastasis in vivo. Transcriptomic (RNA sequencing) pathway analysis revealed alterations in estrogen signaling regulators and pathways, suggesting a role for ZEB2 in endocrine sensitivity in luminal A breast cancer. Expression of ZEB2 was negatively correlated with estrogen receptor complex genes in luminal A patient tumors. Furthermore, treatment with 17ß-estradiol (E2) or the estrogen receptor antagonist ICI 182,780 had no effect on growth of ZEB2-overexpressing cells. CONCLUSION: ZEB2 is a multi-functional regulator of drug sensitivity, cell migration, and metastasis in ER + breast cancer and functions through non-canonical mechanisms.
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Neoplasias de la Mama , Transición Epitelial-Mesenquimal , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal/genética , Femenino , Fulvestrant , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genéticaRESUMEN
This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).
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Perfilación de la Expresión Génica/métodos , ARN Mensajero/genética , Análisis de Secuencia de ARN , Línea Celular Tumoral , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Anotación de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , ARN Ribosómico/química , ARN Ribosómico/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasas/químicaRESUMEN
Drosophila cell lines are used by researchers to investigate various cell biological phenomena. It is crucial to exercise good cell culture practice. Poor handling can lead to both inter- and intra-species cross-contamination. Prolonged culturing can lead to introduction of large- and small-scale genomic changes. These factors, therefore, make it imperative that methods to authenticate Drosophila cell lines are developed to ensure reproducibility. Mammalian cell line authentication is reliant on short tandem repeat (STR) profiling; however, the relatively low STR mutation rate in Drosophila melanogaster at the individual level is likely to preclude the value of this technique. In contrast, transposable elements (TEs) are highly polymorphic among individual flies and abundant in Drosophila cell lines. Therefore, we investigated the utility of TE insertions as markers to discriminate Drosophila cell lines derived from the same or different donor genotypes, divergent sub-lines of the same cell line, and from other insect cell lines. We developed a PCR-based next-generation sequencing protocol to cluster cell lines based on the genome-wide distribution of a limited number of diagnostic TE families. We determined the distribution of five TE families in S2R+, S2-DRSC, S2-DGRC, Kc167, ML-DmBG3-c2, mbn2, CME W1 Cl.8+, and ovarian somatic sheath Drosophila cell lines. Two independent downstream analyses of the next-generation sequencing data yielded similar clustering of these cell lines. Double-blind testing of the protocol reliably identified various Drosophila cell lines. In addition, our data indicate minimal changes with respect to the genome-wide distribution of these five TE families when cells are passaged for at least 50 times. The protocol developed can accurately identify and distinguish the numerous Drosophila cell lines available to the research community, thereby aiding reproducible Drosophila cell culture research.
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Línea Celular , Elementos Transponibles de ADN , Drosophila , Animales , Elementos Transponibles de ADN/genética , Drosophila/genética , Drosophila melanogaster/genética , Genoma de los Insectos , Reproducibilidad de los ResultadosRESUMEN
Pneumonia accounts for over 20% of deaths worldwide in children aged 1 to 5 years, disproportionately affecting lower- and middle-income countries. Effective, highly multivalent pneumococcal vaccines are available to decrease disease burden, with numerous new vaccines currently under development to serve a variety of worldwide markets. However, pneumococcal conjugate vaccines are among the hardest biologics to manufacture and characterize due to their complexity and heterogeneity. Current characterization methods are often inherently singleplex, requiring separate tests for each serotype present. In addition, identity and quantity are often determined with separate methods. We developed the VaxArray pneumococcal assay for applications in identity, quantity, and stability testing of pneumococcal polysaccharide and pneumococcal conjugate vaccines. The VaxArray pneumococcal assay has a time to result of less than 30 min and is an off-the-shelf multiplexed, microarray-based immunoassay kit that can identify and simultaneously quantify 23 pneumococcal polysaccharide serotypes common to many on-market and in-development vaccines. Here, we highlight the potential of the assay for identity testing by showing high reactivity and serotype specificity to a wide variety of native polysaccharides, CRM197-conjugated polysaccharides, and drug product. The assay also has vaccine-relevant lower limits of quantification in the low-to-mid ng/mL range and can be used for accurate quantification even in adjuvanted vaccines. Excellent correlation to the anthrone assay is demonstrated, with VaxArray resulting in significantly improved precision over this antiquated chemical method.
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Measles-containing vaccines (MCV), specifically vaccines against measles and rubella (MR), are extremely effective and critical for the eradication of measles and rubella diseases. In developed countries, vaccination rates are high and vaccines are readily available, but continued high prevalence of both diseases in developing countries and surges in measles deaths in recent years have highlighted the need to expand vaccination efforts. To meet demand for additional vaccines at a globally affordable price, it is highly desirable to streamline vaccine production thereby reducing cost and speeding up time to delivery. MR vaccine characterization currently relies on the 50% cell culture infectious dose (CCID50) assay, an endpoint assay with low reproducibility that requires 10-14 days to complete. For streamlining bioprocess analysis and improving measurement precision relative to CCID50, we developed the VaxArray Measles and Rubella assay kit, which is based on a multiplexed microarray immunoassay with a 5-hour time to result. Here we demonstrate vaccine-relevant sensitivity ranging from 345 to 800 IFU/mL up to 100,000 IFU/mL (infectious units per mL) and specificity that allows simultaneous analysis in bivalent vaccine samples. The assay is sensitive to antigen stability and has minimal interference from common vaccine additives. The assay exhibits high reproducibility and repeatability, with 15% CV, much lower than the typical 0.3 log10 error (â¼65%) observed for the CCID50 assay. The intact protein concentration measured by VaxArray is reasonably correlated to, but not equivalent to, CCID50 infectivity measurements for harvest samples. However, the measured protein concentration exhibits equivalency to CCID50 for more purified samples, including concentrated virus pools and monovalent bulks, making the assay a useful new tool for same-day analysis of vaccine samples for bioprocess development, optimization, and monitoring.
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Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.
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Adenosina Trifosfato/química , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Compuestos de Fenilurea/química , Inhibidores de Proteínas Quinasas/química , Pirazoles/química , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Células HeLa , Humanos , Cinética , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/farmacología , Fosforilación , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/síntesis química , Pirazoles/farmacología , Relación Estructura-ActividadRESUMEN
Neuraminidase (NA) immunity leads to decreased viral shedding and reduced severity of influenza disease; however, NA content in influenza vaccines is currently not regulated, resulting in inconsistent quality and quantity of NA that can vary from manufacturer to manufacturer, from year to year, and from lot to lot. To address this problem, we have developed an assay for NA quantification that could be used by the industry to move toward developing influenza vaccines that induce a predictable immune response to NA. The VaxArray Influenza Seasonal NA Potency Assay (VXI-sNA) is a multiplexed sandwich immunoassay that relies on six subtype-specific monoclonal antibodies printed in microarray format and a suite of fluor-conjugated "label" antibodies. The performance of the assay as applied to a wide range of influenza vaccines is described herein. The assay demonstrated high NA subtype specificity and high sensitivity, with quantification limits ranging from 1 to 60 ng/mL and linear dynamic ranges of 24-500-fold. When compared to an enzymatic activity assay for samples exposed to thermal degradation conditions, the assay was able to track changes in protein stability over time and exhibited good correlation with enzyme activity. The assay also demonstrated excellent analytical precision with relative error ranging from 6 to 12% over day-to-day, user-to-user, and lot-to-lot variation. The high sensitivity and reproducibility of the assay enabled robust detection and quantification of NA in crude in-process samples and low-dose, adjuvanted vaccines with an accuracy of 100 ± 10%.
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The VaxArray Influenza Pandemic HA (VXI-pHA) potency assay is a multiplexed sandwich immunoassay that consists of nine broadly reactive yet subtype-specific monoclonal capture antibodies printed in microarray format and a suite of fluor-labeled secondary antibodies that were selected to probe conserved HA epitopes. VXI-pHA was designed to optimize the probability that the ready-to-use assay would work for the most concerning, emergent influenza A strains, eliminating the need for the time-consuming process of reference reagents production. The performance of this new potency test was evaluated using a panel of 48 potentially pandemic strains of influenza viruses and vaccines spanning 16 years of antigenic drift, including the most recent pre-pandemic vaccine being developed against the "5th wave" A/H7N9 virus. The VXI-pHA assay demonstrated coverage of 93%, 92%, and 100% for H5, H7, and H9 antigens, respectively. The assay demonstrated high sensitivity with linear dynamic ranges of more than 150-fold and quantification limits ranging from 1 to 5 ng/mL. For three production lots of H7N9 monobulk drug substance, the assay exhibited excellent accuracy (100 ± 6%) and analytical precision (CV 6 ± 2%). The high assay sensitivity enabled robust detection and quantification of hemagglutinin in crude in-process samples and low-dose, adjuvanted vaccines with an accuracy of 100 ± 10%.
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microRNAs (miRNAs) are small noncoding RNA molecules involved in the regulation of gene expression and play critical roles in human malignancies. Next-generation sequencing analysis of the MCF-7 breast cancer cell line overexpressing miR-335-5p and miR-335-3p demonstrated that the miRNA duplex repressed genes involved in the ERα signaling pathway, and enhanced resistance of MCF-7 cells to the growth inhibitory effects of tamoxifen. These data suggest that despite its conventional role in tumor suppression, the miR-335 transcript can also play an oncogenic role in promoting agonistic estrogen signaling in a cancerous setting.
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Resistencia a Antineoplásicos/genética , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , MicroARNs/metabolismo , Tamoxifeno/farmacología , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Receptor alfa de Estrógeno/metabolismo , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células MCF-7 , MicroARNs/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
RNA-Seq is the leading technology for analyzing gene expression on a global scale across a broad spectrum of sample types. However, due to chemical modifications by fixation or degradation due to collection methods, samples often contain an abundance of RNA that is no longer intact, and the capability of current RNA-Seq protocols to accurately quantify such samples is often limited. We have developed an RNA-Seq protocol to address these key issues as well as quantify gene expression from the whole transcriptome. Furthermore, for compatibility with improved sequencing platforms, we use restructured adapter sequences to generate libraries for Illumina HiSeq, MiSeq, and NextSeq platforms. Our protocol utilizes duplex-specific nuclease (DSN) to remove abundant ribosomal RNA sequences while retaining other types of RNA for superior transcriptome profiling from low quantity input. We employ the Illumina sequencing platform, but this method is described in sufficient detail to adapt to other platforms.
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Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Mensajero/genética , ARN Ribosómico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transcriptoma , Línea Celular Tumoral , Biblioteca de Genes , Humanos , Neoplasias/genética , Sondas de Oligonucleótidos/química , División del ARN , ARN Bicatenario/química , ARN Bicatenario/genética , ARN Ribosómico/química , Ribonucleasas/química , Análisis de Secuencia de ARNRESUMEN
During embryonic development of the Drosophila brain, the Hox gene labial is required for the regionalized specification of the tritocerebral neuromere. In order to gain further insight into the mechanisms of Hox gene action in the CNS, we have studied the molecular and genetic basis of cross-regulatory interactions between labial and other more posterior Hox genes using the GAL4/UAS system for targeted misexpression. Misexpression of posterior Hox genes in the embryonic neuroectoderm results in a labial loss-of function phenotype and a corresponding lack of Labial protein expression in the tritocerebrum. This is due to repression of labial gene transcription in the embryonic brain. Enhancer analysis suggests that this transcriptional repression operates on a 3.65 kb brain-specific labial-enhancer element. A functional analysis of Antennapedia and Ultrabithorax protein domains shows that the transcriptional repression of labial requires homeodomain-DNA interactions but is not dependent on a functional hexapeptide. The repressive activity of a Hox protein on labial expression in the tritocerebrum can, however, be abolished by concomitant misexpression of a Hox protein and the cofactors Homothorax and nuclear-targeted Extradenticle. Taken together, these results provide novel and detailed insight into the cross-regulatory interactions of Hox genes in embryonic brain development and suggest that specification of tritocerebral neuronal identity requires equilibrated levels of a Hox protein and Hth and n-Exd cofactors.
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Encéfalo/embriología , Proteínas de Drosophila/fisiología , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Proteína con Homeodominio Antennapedia , Encéfalo/metabolismo , Sistema Nervioso Central , Cruzamientos Genéticos , Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/biosíntesis , Drosophila melanogaster , Desarrollo Embrionario/fisiología , Elementos de Facilitación Genéticos , Proteínas de Homeodominio/biosíntesis , Inmunohistoquímica , Péptidos/química , Fenotipo , Unión Proteica , Factores de Tiempo , Distribución Tisular , Factores de Transcripción/biosíntesis , Transcripción GenéticaRESUMEN
BACKGROUND: Although the global use of the endocrine-disrupting chemical DDT has decreased, its persistence in the environment has resulted in continued human exposure. Accumulating evidence suggests that DDT exposure has long-term adverse effects on development, yet the impact on growth and differentiation of adult stem cells remains unclear. OBJECTIVES: Human mesenchymal stem cells (MSCs) exposed to DDT were used to evaluate the impact on stem cell biology. METHODS: We assessed DDT-treated MSCs for self-renewal, proliferation, and differentiation potential. Whole genome RNA sequencing was performed to assess gene expression in DDT-treated MSCs. RESULTS: MSCs exposed to DDT formed fewer colonies, suggesting a reduction in self-renewal potential. DDT enhanced both adipogenic and osteogenic differentiation, which was confirmed by increased mRNA expression of glucose transporter type 4 (GLUT4), lipoprotein lipase (LpL), peroxisome proliferator-activated receptor gamma (PPARγ), leptin, osteonectin, core binding factor 1 (CBFA1), and FBJ murine osteosarcoma viral oncogene homolog (c-Fos). Expression of factors in DDT-treated cells was similar to that in estrogen-treated MSCs, suggesting that DDT may function via the estrogen receptor (ER)-mediated pathway. The coadministration of ICI 182,780 blocked the effects of DDT. RNA sequencing revealed 121 genes and noncoding RNAs to be differentially expressed in DDT-treated MSCs compared with controls cells. CONCLUSION: Human MSCs provide a powerful biological system to investigate and identify the molecular mechanisms underlying the effects of environmental agents on stem cells and human health. MSCs exposed to DDT demonstrated profound alterations in self-renewal, proliferation, differentiation, and gene expression, which may partially explain the homeostatic imbalance and increased cancer incidence among those exposed to long-term EDCs.
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Adipogénesis/efectos de los fármacos , DDT/toxicidad , Disruptores Endocrinos/toxicidad , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular , Receptor alfa de Estrógeno , Expresión Génica , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , PPAR gamma , Receptores de Estrógenos , Análisis de Secuencia de ARNRESUMEN
Stranded whole transcriptome RNA-Seq described in this unit captures quantitative expression data for all types of RNA including, but not limited to, miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (large non-coding intergenic RNA), SRP RNA (signal recognition particle RNA), tRNA (transfer RNA), mtRNA (mitochondrial RNA), and mRNA (messenger RNA). The size and nature of these types of RNA are irrelevant to the approach described here. Barcoded libraries for multiplexing on the Illumina platform are generated with this approach but it can be applied to other platforms with a few modifications.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Largo no Codificante/análisis , ARN Mensajero/análisis , ARN Pequeño no Traducido/análisis , ARN de Transferencia/análisis , ARN/análisis , Transcriptoma , ADN Complementario/análisis , ADN Complementario/genética , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Humanos , Reacción en Cadena de la Polimerasa/métodos , ARN/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mitocondrial , ARN Pequeño no Traducido/clasificación , ARN Pequeño no Traducido/genética , ARN de Transferencia/genética , Transcripción ReversaRESUMEN
Epithelial to mesenchymal transition (EMT) involves loss of an epithelial phenotype and activation of a mesenchymal one. Enhanced expression of genes associated with a mesenchymal transition includes ZEB1/2, TWIST, and FOXC1. miRNAs are known regulators of gene expression and altered miRNA expression is known to enhance EMT in breast cancer. Here we demonstrate that the tumor suppressive miRNA family, miR-200, is not expressed in triple negative breast cancer (TNBC) cell lines and that miR-200b-3p over-expression represses EMT, which is evident through decreased migration and increased CDH1 expression. Despite the loss of migratory capacity following re-expression of miR-200b-3p, no subsequent loss of the conventional miR-200 family targets and EMT markers ZEB1/2 was observed. Next generation RNA-sequencing analysis showed that enhanced expression of pri-miR-200b lead to ectopic expression of both miR-200b-3p and miR-200b-5p with multiple isomiRs expressed for each of these miRNAs. Furthermore, miR-200b-5p was expressed in the receptor positive, epithelial breast cancer cell lines but not in the TNBC (mesenchymal) cell lines. In addition, a compensatory mechanism for miR-200b-3p/200b-5p targeting, where both miRNAs target the RHOGDI pathway leading to non-canonical repression of EMT, was demonstrated. Collectively, these data are the first to demonstrate dual targeting by miR-200b-3p and miR-200b-5p and a previously undescribed role for microRNA processing and strand expression in EMT and TNBC, the most aggressive breast cancer subtype.
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Transición Epitelial-Mesenquimal/genética , MicroARNs/genética , Neoplasias de la Mama Triple Negativas/genética , Inhibidor alfa de Disociación del Nucleótido Guanina rho/metabolismo , Antígenos CD , Secuencia de Bases , Cadherinas/biosíntesis , Línea Celular Tumoral , Movimiento Celular/genética , Femenino , Factores de Transcripción Forkhead/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Células MCF-7 , MicroARNs/biosíntesis , Proteínas Nucleares/genética , Proteínas Represoras/genética , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Proteína 1 Relacionada con Twist/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Here we describe how to generate customized microinjection needles from glass capillary tubes. Controls demonstrate the range of variables and effects on needle tip shape using a standard Flaming/Brown micropipet needle puller. Needles generated with two-cycle pulls provide a wider range of needle shapes in a predictable fashion. We used the needle puller's ramp function for multiple-cycle programs to determine the useful range of heat settings inherent to the glass capillary tube. This articlefocuses primarily on the preparation of injection needles utilized for P-element-mediated germ-line transformation in Drosophila melanogaster that do not require the dechorionation of the egg. However, these types of needles can be usefulfor numerous other types of injections, such as RNA interference, homologous recombination mutagenesis, morpholinos, transient gene regulation, drug delivery, and the transfer of cytoplasmic factors that are useful in a wide range of biological systems ranging from plants to vertebrates. Using our standard needle, we correlate the survival of injected D. melanogaster embryos with transformation efficiencies and plasmid construct characteristics.
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Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Técnicas de Transferencia de Gen/instrumentación , Vidrio , Microinyecciones/instrumentación , Agujas , Presión del Aire , Animales , Drosophila melanogaster/embriología , Diseño de Equipo , Células Germinativas , Calor , Microinyecciones/métodos , Tamaño de la Partícula , Plásmidos , Control de Calidad , Recombinación Genética , Sensibilidad y Especificidad , Transformación Genética , ViscosidadRESUMEN
PURPOSE: To investigate SGI-110 as a "chemosensitizer" in ovarian cancer and to assess its effects on tumor suppressor genes (TSG) and chemoresponsiveness-associated genes silenced by DNA methylation in ovarian cancer. EXPERIMENTAL DESIGN: Several ovarian cancer cell lines were used for in vitro and in vivo platinum resensitization studies. Changes in DNA methylation and expression levels of TSG and other cancer-related genes in response to SGI-110 were measured by pyrosequencing and RT-PCR. RESULTS: We demonstrate in vitro that SGI-110 resensitized a range of platinum-resistant ovarian cancer cells to cisplatin (CDDP) and induced significant demethylation and reexpression of TSG, differentiation-associated genes, and putative drivers of ovarian cancer cisplatin resistance. In vivo, SGI-110 alone or in combination with CDDP was well tolerated and induced antitumor effects in ovarian cancer xenografts. Pyrosequencing analyses confirmed that SGI-110 caused both global (LINE1) and gene-specific hypomethylation in vivo, including TSGs (RASSF1A), proposed drivers of ovarian cancer cisplatin resistance (MLH1 and ZIC1), differentiation-associated genes (HOXA10 and HOXA11), and transcription factors (STAT5B). Furthermore, DNA damage induced by CDDP in ovarian cancer cells was increased by SGI-110, as measured by inductively coupled plasma-mass spectrometry analysis of DNA adduct formation and repair of cisplatin-induced DNA damage. CONCLUSIONS: These results strongly support further investigation of hypomethylating strategies in platinum-resistant ovarian cancer. Specifically, SGI-110 in combination with conventional and/or targeted therapeutics warrants further development in this setting.
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Azacitidina/análogos & derivados , Metilación de ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Azacitidina/administración & dosificación , Azacitidina/farmacología , Línea Celular Tumoral , Cisplatino/administración & dosificación , Cisplatino/farmacología , Aductos de ADN , Modelos Animales de Enfermedad , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Histonas/metabolismo , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ovarian cancer (OC) is most often contained within the peritoneal cavity, making it an ideal disease for adenoviral-delivered gene therapies. In effort to develop a safe and effective gene therapy for OC, we created a replication deficient adenovirus bearing the herpes simplex thymidine kinase (HSV-tk) gene under direction of the tumor specific promoter human epididymis protein 4 (HE4). The purpose of this study was to investigate the ability of our adenoviral construct to transduce OC cells in vitro and mediate transgene expression of HSV-tk, thereby sensitizing OC to the pro-drug ganciclovir. Cisplatin-sensitive (CS) and -resistant (CR) A2780 OC cells, infected with virus for 6 hours at 100, 500, and 1000 multiplicity of infection followed by ganciclovir treatment every other day for 5 days, were assayed for cell viability. Adenoviral-mediated transgene expression increased with increasing amounts of virus and peaked at 48 hours after transduction in both A2780-CS and -CR. Unexpectedly, ganciclovir alone was slightly toxic to both A2780 cell lines (IC50 of 234.9 µg/mL and 257.2 µg/mL in A2780-CS and -CR, respectively). Transduction with ADV-HE4-HSV-tk followed by ganciclovir treatment increased (P<0.05) cell killing up to ten-fold, lowering the IC50 to 23.9 µg/mL and 32.6 µg/mL in A2780-CS and -CR, respectively, at 1000 multiplicity of infection. The results support the potential use of this approach as a gene therapy for OC, a disease that accounts for more deaths than any other cancer of the female reproductive system.
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Multidimensional separations combined with mass spectrometry are used to study the proteins that are present in two states of Drosophila melanogaster: the whole embryo and the adult head. The approach includes the incorporation of a gas-phase separation dimension in which ions are dispersed according to differences in their mobilities and is described as a means of providing a detailed analytical map of the proteins that are present. Overall, we find evidence for 1133 unique proteins. In total, 780 are identified in the head, and 660 are identified in the embryo. Only 307 proteins are in common to both developmental stages, indicating that there are significant differences in these proteomes. A comparison of the proteome to a database of mRNAs that are found from analysis by cDNA approaches (i.e., transcriptome) also shows little overlap. All of this information is discussed in terms of the relationship between the predicted genome, and measured transcriptomes and proteomes. Additionally, the merits and weaknesses of current technologies are assessed in some detail.
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Cromatografía Liquida/métodos , Drosophila melanogaster , Proteínas de Insectos , Espectrometría de Masas/métodos , Proteoma/análisis , Animales , Calibración , Cromatografía Liquida/instrumentación , Bases de Datos de Ácidos Nucleicos , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genoma , Proteínas de Insectos/genética , Proteínas de Insectos/aislamiento & purificación , Proteínas de Insectos/metabolismo , Espectrometría de Masas/instrumentación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Reproducibilidad de los ResultadosRESUMEN
The proteomes of three heads of individual Drosophila melanogaster organisms have been analyzed and compared by a combination of liquid chromatography, ion mobility spectrometry, and mass spectrometry approaches. In total, 197 proteins are identified among all three individuals (an average of 120 +/- 20 proteins per individual), of which at least 101 proteins are present in all three individuals. Within all three datasets, more than 25 000 molecular ions (an average of 9000 +/- 2000 per individual) corresponding to protonated precursor ions of individual peptides have been observed. A comparison of peaks among the datasets reveals that peaks corresponding to protonated peptides that are found in all heads are more intense than those features that appear between pairs of or within only one of the individuals. Moreover, there is little variability in the relative intensities of the peaks common among all individuals. It appears that it is the lower abundance components of the proteome that play the most significant role in determining unique features of individuals.
Asunto(s)
Cromatografía Liquida/métodos , Drosophila melanogaster/anatomía & histología , Proteínas de Insectos/análisis , Espectrometría de Masas/métodos , Proteoma/análisis , Animales , Proteínas de Insectos/genética , Iones/análisis , Datos de Secuencia MolecularRESUMEN
A linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome.