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1.
J Pathol ; 2020 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-33135777

RESUMEN

The practical application of genome-scale technologies to precision oncology research requires flexible tissue processing strategies that can be used to differentially select both tumour and normal cell populations from formalin-fixed, paraffin-embedded tissues. As tumour sequencing scales towards clinical implementation, practical difficulties in scheduling and obtaining fresh tissue biopsies at scale, including blood samples as surrogates for matched 'normal' DNA, have focused attention on the use of formalin-preserved clinical samples collected routinely for diagnostic purposes. In practice, such samples often contain both tumour and normal cells which, if correctly partitioned, could be used to profile both tumour and normal genomes, thus identifying somatic alterations. Here we report a semi-automated method for laser microdissecting entire slide-mounted tissue sections to enrich for cells of interest with sufficient yield for whole genome and transcriptome sequencing. Using this method, we demonstrated enrichment of tumour material from mixed tumour-normal samples by up to 67%. Leveraging new methods that allow for the extraction of high-quality nucleic acids from small amounts of formalin-fixed tissues, we further showed that the method was successful in yielding sequence data of sufficient quality for use in BC Cancer's Personalized OncoGenomics (POG) program. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

2.
PLoS One ; 14(10): e0224578, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31671154

RESUMEN

Next generation RNA-sequencing (RNA-seq) is a flexible approach that can be applied to a range of applications including global quantification of transcript expression, the characterization of RNA structure such as splicing patterns and profiling of expressed mutations. Many RNA-seq protocols require up to microgram levels of total RNA input amounts to generate high quality data, and thus remain impractical for the limited starting material amounts typically obtained from rare cell populations, such as those from early developmental stages or from laser micro-dissected clinical samples. Here, we present an assessment of the contemporary ribosomal RNA depletion-based protocols, and identify those that are suitable for inputs as low as 1-10 ng of intact total RNA and 100-500 ng of partially degraded RNA from formalin-fixed paraffin-embedded tissues.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Ribosómico/genética , Análisis de Secuencia de ARN/métodos , Animales , Secuencia de Bases/genética , Perfilación de la Expresión Génica/métodos , Humanos , Mamíferos/genética , ARN/genética , ARN Mensajero/genética , Fijación del Tejido/métodos , Transcriptoma/genética
3.
Sci Rep ; 9(1): 12744, 2019 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-31484940

RESUMEN

Crystalline materials exhibit long-range ordered lattice unit, within which resides nonperiodic structural features called defects. These crystallographic defects play a vital role in determining the physical and mechanical properties of a wide range of material systems. While computer vision has demonstrated success in recognizing feature patterns in images with well-defined contrast, automated identification of nanometer scale crystallographic defects in electron micrographs governed by complex contrast mechanisms is still a challenging task. Here, building upon an advanced defect imaging mode that offers high feature clarity, we introduce DefectSegNet - a new convolutional neural network (CNN) architecture that performs semantic segmentation of three common crystallographic defects in structural alloys: dislocation lines, precipitates and voids. Results from supervised training on a small set of high-quality defect images of steels show high pixel-wise accuracy across all three types of defects: 91.60 ± 1.77% on dislocations, 93.39 ± 1.00% on precipitates, and 98.85 ± 0.56% on voids. We discuss the sources of uncertainties in CNN prediction and the training data in terms of feature density, representation and homogeneity and their effects on deep learning performance. Further defect quantification using DefectSegNet prediction outperforms human expert average, presenting a promising new workflow for fast and statistically meaningful quantification of materials defects.

4.
Biotechniques ; 66(2): 85-92, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30744412

RESUMEN

The analysis of cell-free circulating tumor DNA (ctDNA) is potentially a less invasive, more dynamic assessment of cancer progression and treatment response than characterizing solid tumor biopsies. Standard isolation methods require separation of plasma by centrifugation, a time-consuming step that complicates automation. To address these limitations, we present an automatable magnetic bead-based ctDNA isolation method that eliminates centrifugation to purify ctDNA directly from peripheral blood (PB). To develop and test our method, ctDNA from cancer patients was purified from PB and plasma. We found that allelic fractions of somatic single-nucleotide variants from target gene capture libraries were comparable, indicating that the PB ctDNA purification method may be a suitable replacement for the plasma-based protocols currently in use.


Asunto(s)
Ácidos Nucleicos Libres de Células/sangre , ADN Tumoral Circulante/sangre , Ensayos Analíticos de Alto Rendimiento/métodos , Neoplasias/sangre , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/aislamiento & purificación , Ácidos Nucleicos Libres de Células/aislamiento & purificación , ADN Tumoral Circulante/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Neoplasias/genética
5.
Nucleic Acids Res ; 47(2): e12, 2019 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-30418619

RESUMEN

Tissues used in pathology laboratories are typically stored in the form of formalin-fixed, paraffin-embedded (FFPE) samples. One important consideration in repurposing FFPE material for next generation sequencing (NGS) analysis is the sequencing artifacts that can arise from the significant damage to nucleic acids due to treatment with formalin, storage at room temperature and extraction. One such class of artifacts consists of chimeric reads that appear to be derived from non-contiguous portions of the genome. Here, we show that a major proportion of such chimeric reads align to both the 'Watson' and 'Crick' strands of the reference genome. We refer to these as strand-split artifact reads (SSARs). This study provides a conceptual framework for the mechanistic basis of the genesis of SSARs and other chimeric artifacts along with supporting experimental evidence, which have led to approaches to reduce the levels of such artifacts. We demonstrate that one of these approaches, involving S1 nuclease-mediated removal of single-stranded fragments and overhangs, also reduces sequence bias, base error rates, and false positive detection of copy number and single nucleotide variants. Finally, we describe an analytical approach for quantifying SSARs from NGS data.


Asunto(s)
Artefactos , Fijadores , Formaldehído , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN , Animales , Biblioteca Genómica , Genómica , Calor , Ratones Endogámicos C57BL , Adhesión en Parafina
6.
BMC Genomics ; 18(1): 515, 2017 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-28679365

RESUMEN

BACKGROUND: RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming. METHODS: Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. RESULTS: This work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments. CONCLUSIONS: These developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.


Asunto(s)
Biblioteca de Genes , ARN Mensajero , Análisis de Secuencia de ARN/métodos , Manejo de Especímenes/normas , Células HL-60 , Humanos
7.
PLoS One ; 12(6): e0178706, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28570594

RESUMEN

Curation and storage of formalin-fixed, paraffin-embedded (FFPE) samples are standard procedures in hospital pathology laboratories around the world. Many thousands of such samples exist and could be used for next generation sequencing analysis. Retrospective analyses of such samples are important for identifying molecular correlates of carcinogenesis, treatment history and disease outcomes. Two major hurdles in using FFPE material for sequencing are the damaged nature of the nucleic acids and the labor-intensive nature of nucleic acid purification. These limitations and a number of other issues that span multiple steps from nucleic acid purification to library construction are addressed here. We optimized and automated a 96-well magnetic bead-based extraction protocol that can be scaled to large cohorts and is compatible with automation. Using sets of 32 and 91 individual FFPE samples respectively, we generated libraries from 100 ng of total RNA and DNA starting amounts with 95-100% success rate. The use of the resulting RNA in micro-RNA sequencing was also demonstrated. In addition to offering the potential of scalability and rapid throughput, the yield obtained with lower input requirements makes these methods applicable to clinical samples where tissue abundance is limiting.


Asunto(s)
Automatización , ADN/aislamiento & purificación , Formaldehído/química , Secuenciación de Nucleótidos de Alto Rendimiento , Adhesión en Parafina , ARN/aislamiento & purificación , Fijación del Tejido/métodos , ADN/genética , ARN/genética
8.
Clin Cancer Res ; 22(17): 4466-77, 2016 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-27140928

RESUMEN

PURPOSE: Persistent androgen receptor (AR) transcriptional activity is clinically evident in castration-resistant prostate cancer (CRPC). Therefore, AR remains as a viable therapeutic target for CRPC. All current hormonal therapies target the C-terminus ligand-binding domain (LBD) of AR. By using EPI to target AR activation function-1 (AF-1), in the N-terminal domain that is essential for AR transactivation, we evaluate the ability of EPI to overcome several clinically relevant AR-related mechanisms of resistance. EXPERIMENTAL DESIGN: To study the effect of EPI on AR transcriptional activity against overexpressed coactivators, such as SRC1-3 and p300, luciferase reporter assays were performed using LNCaP cells. AR-negative COS-1 cells were employed for reporter assays to examine whether the length of polyglutamine tract affects inhibition by EPI. The effect of EPI on constitutively active AR splice variants was studied in LNCaP95 cells, which express AR-V7 variant. To evaluate the effect of EPI on the proliferation of LNCaP95 cells, we performed in vitro BrdUrd incorporation assay and in vivo studies using xenografts in mice. RESULTS: EPI effectively overcame several molecular alterations underlying aberrant AR activity, including overexpressed coactivators, AR gain-of-function mutations, and constitutively active AR-V7. EPI inhibited AR transcriptional activity regardless of the length of polyglutamine tract. Importantly, EPI significantly inhibited the in vitro and in vivo proliferation of LNCaP95 prostate cancer cells, which are androgen independent and enzalutamide resistant. CONCLUSIONS: These findings support EPI as a promising therapeutic agent to treat CRPC, particularly against tumors driven by constitutively active AR splice variants that are resistant to LBD-targeting drugs. Clin Cancer Res; 22(17); 4466-77. ©2016 AACRSee related commentary by Sharp et al., p. 4280.


Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Compuestos de Bencidrilo/farmacología , Clorhidrinas/farmacología , Resistencia a Antineoplásicos , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Receptores Androgénicos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Mutación , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Unión Proteica , Empalme del ARN , Receptores Androgénicos/genética , Transducción de Señal/efectos de los fármacos , Activación Transcripcional , Ensayos Antitumor por Modelo de Xenoinjerto
9.
J Clin Invest ; 123(7): 2948-60, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23722902

RESUMEN

Hormone therapies for advanced prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD), but these ultimately fail and the disease progresses to lethal castration-resistant prostate cancer (CRPC). The mechanisms that drive CRPC are incompletely understood, but may involve constitutively active AR splice variants that lack the LBD. The AR N-terminal domain (NTD) is essential for AR activity, but targeting this domain with small-molecule inhibitors is complicated by its intrinsic disorder. Here we investigated EPI-001, a small-molecule antagonist of AR NTD that inhibits protein-protein interactions necessary for AR transcriptional activity. We found that EPI analogs covalently bound the NTD to block transcriptional activity of AR and its splice variants and reduced the growth of CRPC xenografts. These findings suggest that the development of small-molecule inhibitors that bind covalently to intrinsically disordered proteins is a promising strategy for development of specific and effective anticancer agents.


Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Antineoplásicos Hormonales/farmacología , Compuestos de Bencidrilo/farmacología , Clorhidrinas/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Antagonistas de Receptores Androgénicos/química , Animales , Antineoplásicos Hormonales/química , Compuestos de Bencidrilo/química , Células COS , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Clorhidrinas/química , Química Clic , Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Orquiectomía , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Unión Proteica , Estructura Terciaria de Proteína , Receptores Androgénicos/química , Receptores Androgénicos/genética , Estereoisomerismo , Activación Transcripcional/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
10.
mBio ; 3(6)2012 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-23149485

RESUMEN

UNLABELLED: The Toxoplasma gondii SRS gene superfamily is structurally related to SRS29B (formerly SAG1), a surface adhesin that binds host cells and stimulates host immunity. Comparative genomic analyses of three Toxoplasma strains identified 182 SRS genes distributed across 14 chromosomes at 57 genomic loci. Eight distinct SRS subfamilies were resolved. A core 69 functional gene orthologs were identified, and strain-specific expansions and pseudogenization were common. Gene expression profiling demonstrated differential expression of SRS genes in a developmental-stage- and strain-specific fashion and identified nine SRS genes as priority targets for gene deletion among the tissue-encysting coccidia. A Δsag1 sag2A mutant was significantly attenuated in murine acute virulence and showed upregulated SRS29C (formerly SRS2) expression. Transgenic overexpression of SRS29C in the virulent RH parent was similarly attenuated. Together, these findings reveal SRS29C to be an important regulator of acute virulence in mice and demonstrate the power of integrated genomic analysis to guide experimental investigations. IMPORTANCE: Parasitic species employ large gene families to subvert host immunity to enable pathogen colonization and cause disease. Toxoplasma gondii contains a large surface coat gene superfamily that encodes adhesins and virulence factors that facilitate infection in susceptible hosts. We generated an integrated bioinformatic resource to predict which genes from within this 182-gene superfamily of adhesin-encoding genes play an essential role in the host-pathogen interaction. Targeted gene deletion experiments with predicted candidate surface antigens identified SRS29C as an important negative regulator of acute virulence in murine models of Toxoplasma infection. Our integrated computational and experimental approach provides a comprehensive framework, or road map, for the assembly and discovery of additional key pathogenesis genes contained within other large surface coat gene superfamilies from a broad array of eukaryotic pathogens.


Asunto(s)
Biología Computacional/métodos , Proteínas Protozoarias/genética , Eliminación de Secuencia , Toxoplasma/genética , Toxoplasma/patogenicidad , Factores de Transcripción/genética , Factores de Virulencia/biosíntesis , Animales , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Ratones , Toxoplasmosis Animal/parasitología , Virulencia
11.
PLoS One ; 6(9): e24197, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21909421

RESUMEN

Androgen receptor (AR) is a member of the nuclear receptor family of transcription factors. Upon binding to androgens, AR becomes transcriptionally active to regulate the expression of target genes that harbor androgen response elements (AREs) in their promoters and/or enhancers. AR is essential for the growth and survival of prostate cancer cells and is therefore a target for current and next-generation therapeutic modalities against prostate cancer. Pathophysiologically relevant protein-protein interaction networks involving AR are, however, poorly understood. In this study, we identified the protein FUsed/Translocated in LipoSarcoma (FUS/TLS) as an AR-interacting protein by co-immunoprecipitation of endogenous proteins in LNCaP human prostate cancer cells. The hormonal response of FUS expression in LNCaP cells was shown to resemble that of other AR co-activators. FUS displayed a strong intrinsic transactivation capacity in prostate cancer cells when tethered to basal promoters using the GAL4 system. Chromatin immunoprecipitation experiments showed that FUS was recruited to ARE III of the enhancer region of the PSA gene. Data from ectopic overexpression and "knock-down" approaches demonstrated that AR transcriptional activity was enhanced by FUS. Depletion of FUS reduced androgen-dependent proliferation of LNCaP cells. Thus, FUS is a novel co-activator of AR in prostate cancer cells.


Asunto(s)
Neoplasias de la Próstata/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Receptores Androgénicos/metabolismo , Transactivadores/metabolismo , Secuencia de Aminoácidos , Andrógenos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Espectrometría de Masas , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Transporte de Proteínas/efectos de los fármacos , Proteína FUS de Unión a ARN/química , Receptores Androgénicos/genética , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Transactivadores/química , Activación Transcripcional/efectos de los fármacos
12.
Cell Mol Life Sci ; 68(24): 3971-81, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21748469

RESUMEN

Androgen receptor (AR) is a transcription factor that becomes active upon binding to androgens via its ligand-binding domain (LBD) or in response to signaling cascades initiated by growth factors and cytokines. The activity of AR requires regions within the N-terminal domain (NTD) in a manner that is distinct from the activation of related steroid hormone receptors. Unequivocal evidence has been amassed to consider that the AR axis is the most critical pathway for the progression of prostate cancer. Qualitatively distinct insights into AR pathobiology have been garnered including that AR-regulated gene expression is stage-specifically modulated during disease progression and that the ligand requirement for AR activity could be rendered dispensable because of the expression of constitutively active AR splice variants that are devoid of LBD. The recent appreciation of the clinical challenge that stems from non-gonadal androgens that are not inhibited by traditional hormonal therapies has been tangibly translated into the development of more potent drugs that can potentially lead towards achieving an androgen-free environment. The pre-clinical evidence that proves that AR NTD is a druggable target also forecasts a further paradigm shift in the management of advanced prostate cancer. These advancements together with the identification of more robust AR antagonists and their promising clinical outcome have renewed the hope that targeting the AR pathway remains a sound strategy in the clinical management of prostate cancer. Here, we address these developments with a greater emphasis on the rapidly growing literature on AR splice variants.


Asunto(s)
Empalme Alternativo , Neoplasias de la Próstata/genética , Receptores Androgénicos/fisiología , Antineoplásicos/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Modelos Biológicos , Neoplasias de la Próstata/tratamiento farmacológico , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Receptores Androgénicos/análisis , Receptores Androgénicos/química , Receptores Androgénicos/genética
13.
Cancer Cell ; 17(6): 535-46, 2010 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-20541699

RESUMEN

Castration-recurrent prostate cancer (CRPC) is suspected to depend on androgen receptor (AR). The AF-1 region in the amino-terminal domain (NTD) of AR contains most, if not all, of the transcriptional activity. Here we identify EPI-001, a small molecule that blocked transactivation of the NTD and was specific for inhibition of AR without attenuating transcriptional activities of related steroid receptors. EPI-001 interacted with the AF-1 region, inhibited protein-protein interactions with AR, and reduced AR interaction with androgen-response elements on target genes. Importantly, EPI-001 blocked androgen-induced proliferation and caused cytoreduction of CRPC in xenografts dependent on AR for growth and survival without causing toxicity.


Asunto(s)
Antagonistas de Receptores Androgénicos , Antineoplásicos Hormonales/uso terapéutico , Compuestos de Bencidrilo/uso terapéutico , Castración , Clorhidrinas/uso terapéutico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Andrógenos/farmacología , Animales , Antineoplásicos Hormonales/efectos adversos , Antineoplásicos Hormonales/farmacología , Apoptosis/efectos de los fármacos , Compuestos de Bencidrilo/efectos adversos , Compuestos de Bencidrilo/farmacología , Proteína de Unión a CREB/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Clorhidrinas/efectos adversos , Clorhidrinas/farmacología , ADN/genética , ADN/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Estructura Molecular , Recurrencia Local de Neoplasia/patología , Próstata/anatomía & histología , Próstata/efectos de los fármacos , Próstata/patología , Antígeno Prostático Específico/sangre , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Conformación Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Receptores Androgénicos/metabolismo , Receptores de Esteroides/efectos de los fármacos , Elementos de Respuesta/genética , Serina Endopeptidasas/genética , Activación Transcripcional/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
14.
Mol Biochem Parasitol ; 172(2): 99-106, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20363263

RESUMEN

Capping of mRNAs is strictly coupled to RNA polymerase II transcription and there is evidence, mainly from metazoans, that other steps in pre-mRNA processing show a similar linkage. In trypanosomes, however, the mRNA cap is supplied by a trans spliced leader sequence. Thus pre-mRNAs transcribed by RNA Polymerase I are capped by trans splicing, and translation-competent transgenic mRNAs can be produced by RNA Polymerase I and T7 RNA polymerase so long as the primary transcript has a splice acceptor signal. We quantified the efficiency of processing of trypanosome pre-mRNAs produced from a plasmid integrated either at the tubulin locus, or in an rRNA spacer, and transcribed by RNA polymerase II, RNA polymerase I or T7 RNA polymerase. The processing efficiencies were similar for primary transcripts from the tubulin locus, produced by RNA polymerase II, and for RNA from an rRNA spacer, transcribed by RNA polymerase I. Primary transcripts produced by T7 RNA polymerase from the tubulin locus were processed almost as well. There was therefore no evidence for recruitment of the 3'-splicing apparatus by the RNA polymerase. Abundant transcripts transcribed from the rRNA locus by T7 RNA polymerase were somewhat less efficiently processed.


Asunto(s)
Fosfoglicerato Quinasa/genética , Proteínas Protozoarias/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismo , ARN Protozoario/metabolismo , Trans-Empalme , Trypanosoma brucei brucei/metabolismo , Transcripción Genética , Trypanosoma brucei brucei/enzimología
15.
Am J Pathol ; 175(6): 2264-76, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19893039

RESUMEN

Levels of 27 transcripts were investigated as potential novel markers for prostate cancer, including genes encoding plasma membrane proteins (ADAM2, ELOVL5, MARCKSL1, RAMP1, TMEM30A, and TMEM66); secreted proteins (SPON2, TMEM30A, TMEM66, and truncated TMEFF2 (called POP4)); intracellular proteins (CAMK2N1, DHCR24, GLO1, NGFRAP1, PGK1, PSMA7, SBDS, and YWHAQ); and noncoding transcripts (POP1 (100 kb) from mRNA AK000023), POP2 (4 kb from mRNA AL832227), POP3 (50 kb from EST CFI40309), POP5 (intron of NCAM2, accession DO668384), POP6 (intron of FHIT), POP7 (intron of TNFAIP8), POP8 (intron of EFNA5), POP9 (intron of DSTN), POP10 (intron of ADAM2, accession DO668396), POP11 (87kb from EST BG194644), and POP12 (intron of EST BQ226050)). Expression of POP3 was prostate specific, whereas ADAM2, POP1, POP4, POP10, ELOVL5, RAMP1, and SPON2 had limited tissue expression. ELOVL5, MARCKSL1, NGFRAP1, PGK1, POP2, POP5, POP8, PSMA7, RAMP1, and SPON2 were significantly differentially expressed between laser microdissected malignant versus benign clinical samples of prostate tissue. PGK1, POP2, and POP12 correlated to clinical parameters. Levels of CAMK2N1, GLO1, SDBS, and TMEM30A transcripts tended to be increased in primary prostate cancer from patients who later had biochemical failure. Expression of GLO1, DHCR24, NGFRAP1, KLK3, and RAMP1 were significantly decreased in metastatic castration-recurrent disease compared with androgen-dependent primary prostate cancer. These novel potential biomarkers may therefore be useful in the diagnosis/prognosis of prostate cancer.


Asunto(s)
Biomarcadores de Tumor/análisis , Perfilación de la Expresión Génica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Anciano , Western Blotting , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Rayos Láser , Masculino , Microdisección , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/metabolismo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
16.
Cancer Res ; 69(8): 3433-42, 2009 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-19351846

RESUMEN

Identification of gene expression signatures associated with metastases provides a tool to discern mechanisms and potential therapeutic targets and may lead toward a molecular classification system in pathology. Prostate cancer (CaP) frequently metastasizes to the bone to form osteoblastic lesions. Correlative clinical data and in vitro evidence have led to the hypothesis that osteoblast-derived factors promote hormonal progression of CaP cells. Here, the gene expression signature of CaP exposed to osteoblast-derived factors was identified. This signature included known androgen-regulated genes, oncogenes, tumor suppressors, and genes whose products are involved in apoptosis and cell cycle. A comparative functional genomic approach involved the application of this responsive gene expression signature to clinical samples of human CaP, melanomas, and oral cancers. Cluster analysis revealed that this gene expression signature had specificity for CaP and could resolve clinical specimens according to stage (benign, localized, and metastatic) and androgen sensitivity with an accuracy of 100% and 80%, respectively. Together, these results suggest that factors derived from osteoblasts induce a more advanced phenotype of CaP and promotes hormonal progression.


Asunto(s)
Neoplasias Óseas/genética , Neoplasias Hormono-Dependientes/genética , Osteoblastos/metabolismo , Neoplasias de la Próstata/genética , Animales , Neoplasias Óseas/secundario , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Osteoblastos/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Extractos de Tejidos/farmacología , Trasplante Heterólogo
17.
Nucleic Acids Res ; 36(5): 1634-44, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18250085

RESUMEN

The life cycle of Leishmania alternates between developmental forms residing within the insect vector (e.g. promastigotes) and the mammalian host (amastigotes). In Leishmania nearly all control of gene expression is post-transcriptional and involves sequences in the 3'-untranslated regions (3'UTRs) of mRNAs. Very little is known as to how these cis-elements regulate RNA turnover and translation rates in trypanosomatids and nothing is known about mRNA degradation mechanisms in Leishmania in particular. Here, we use the amastin mRNA-an amastigote-specific transcript-as a model and show that a approximately 100 nt U-rich element (URE) within its 3'UTR significantly accounts for developmental regulation. RNase-H-RNA blot analysis revealed that a major part of the rapid promastigote-specific degradation of the amastin mRNA is not initiated by deadenylation. This is in contrast to the amastin mRNA in amastigotes and to reporter RNAs lacking the URE, which, in common with most eukaryotic mRNAs studied to-date, are deadenylated before being degraded. Moreover, our analysis did not reveal a role for decapping in the stage-specific degradation of the amastin mRNA. Overall, these results suggest that degradation of the amastin mRNA of Leishmania is likely to be bi-phasic, the first phase being stage-specific and dependent on an unusual URE-mediated pathway of mRNA degradation.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Leishmania infantum/crecimiento & desarrollo , Leishmania infantum/genética , Estabilidad del ARN , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/química , Animales , Antígenos de Protozoos/genética , Leishmania infantum/metabolismo , Poli A/metabolismo , Caperuzas de ARN/metabolismo , Uridina/análisis
18.
Med Hypotheses ; 70(2): 375-7, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-17826000

RESUMEN

Most cancer cells would not result in devastating tumours if it were not for their ability to metastasize. The process of cancer metastasis involves significant cell shape, motility, and adhesive changes of pre-cancerous cells, and the remodelling of the extracellular matrix, as well as cognate properties of neighbouring normal cells. Such changes will be hereafter referred to as "tissue fluidity changes". A number of pathogens are known to disseminate to distant organs from sites of infection within a few days. A compromise on the ability to disseminate rapidly could be deleterious to the pathogen (e.g. the pathogen might be cleared before it reaches immuno-privileged sites within its host). Several ways of dissemination could be envisioned - and some are known to occur - ranging from rather passive such as outgrowth and lysis of tissues, residence in the bloodstream, "hitch-hiking" on migratory cells of the immune and lymphatic systems to an active dissemination process involving tissue fluidity changes similar to those that cancer cells invoke to be able to metastasize. The latter is particularly expected to be an important mechanism for the in vivo dissemination of tissue-dwelling pathogens. The mechanisms behind metastasis can, therefore, be viewed as part of the unifying features between cancer cells and pathogens other than their characteristic high proliferation index (at least in one form in the case of digenetic parasites). The current paper presents a synthesis of the hitherto reported but rather scattered data that broadly reinforce the premise of unifying metastasis processes. The overwhelming research outcome in cancer metastasis might therefore serve as a spring board for facilitating the studies of pathogen metastasis and, importantly, relevant cancer treatment strategies can be adopted to combat infectious diseases.


Asunto(s)
Interacciones Huésped-Patógeno/fisiología , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/fisiopatología , Hipoxia de la Célula/genética , Hipoxia de la Célula/fisiología , Matriz Extracelular/fisiología , Expresión Génica , Humanos , Modelos Biológicos , Metástasis de la Neoplasia/terapia
19.
Mol Biochem Parasitol ; 151(1): 52-8, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17118470

RESUMEN

The exosome, a complex of 3'-exoribonucleases and associated proteins, is involved in the degradation of eukaryotic mRNAs in the cytoplasm, and has RNA processing and quality control functions in the nucleus. In yeast, the nuclear exosome differs from the cytoplasmic one in that it contains an additional non-essential component, Rrp6p. In contrast, a small proportion of human RRP6 has been shown to localise to the cytoplasm as well. When we purified the Trypanosoma brucei exosome from cytosolic extracts we found RRP6, apparently in stoichiometric amounts. We here confirm that RRP6 is in the trypanosome cytoplasm and nucleus. The level of RRP6 was unaffected by depletion of core exosome subunits by RNA interference and over-expression of tagged RRP6 was possible, indicating that RRP6 can be present independent of exosome association.


Asunto(s)
Exorribonucleasas/metabolismo , Membranas Intracelulares/enzimología , Trypanosoma brucei brucei/enzimología , Animales , Línea Celular , Núcleo Celular/enzimología , Citosol/enzimología , Exorribonucleasas/genética , Mutación/genética , Unión Proteica , Interferencia de ARN , Trypanosoma brucei brucei/genética
20.
Curr Opin Microbiol ; 10(6): 569-77, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18177626

RESUMEN

Kinetoplastids branched early from the eukaryotic lineage and include several parasitic protozoan species. Up to several hundred kinetoplastid genes are co-transcribed into polycistronic RNAs and individual mRNAs are resolved by coupled co-transcriptional trans-splicing of a universal splice-leader RNA (SL-RNA) and 3'-end maturation processes. Protein-coding genes lack RNA polymerase II promoters. Consequently, most of gene regulation in these organisms occurs post-transcriptionally. Over the last few years, many more genes that are regulated at the mRNA stability level and a few at the translation level have been reported. Almost all major trypanosome homologues of yeast/mammalian mRNA degradation enzymes have been functionally characterized and major pathways identified. Novel paradigms have also recently emerged: regulated post-transcriptional processing of cytoplasmic RNAs, SL-RNA transcriptional silencing-mediated global stress response, and Leishmania-specific large-scale modulation of post-transcriptional gene expression via inactive degenerated retroelements. Several of these developments have greatly benefited from the recently completed genomic sequences and functional genomic studies.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas Protozoarias/metabolismo , Trypanosomatina/crecimiento & desarrollo , Animales , Proteínas Protozoarias/genética , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , Trypanosomatina/genética , Trypanosomatina/metabolismo
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