RESUMEN
The multicellular tumor spheroid (MCTS) model represents a suitable in vitro model recreating in vivo tumor formation. The aim of this study was to identify differentially expressed genes that could potentially serve as predictive gene markers for MCTS and be involved in the formation of MCTS. Using the suppression subtractive hybridization (SSH) method, we identified ERBB2/HER2-interacting protein (Erbin), Tumor rejection gp96 (Tr-gp96), 12S ribosomal RNA (12S rRNA), ATP synthase, Kruppel-like transcription factor 5 (KLF5), transcription factor-like 5 (TCFL5), and the dual-specificity phosphatase 11 (DUSP11) to be overexpressed in 3-day-old HT-29 colon carcinoma MCTSs compared to HT-29 colon carcinoma cells grown in monolayer. We could also confirm overexpression of these genes in HT-29 MCTSs and in MCTSs formed by the human glioblastoma tumor cell lines U343 MG, U373 MG, and DBTRG 05 MG. Knockdown of KLF5, Erbin, DUSP11, and TCFL5 was effectively achieved after transfection of HT-29 cells with the appropriate short-interfering RNAs (siRNAs), and correlated with a significant inhibition of MCTS formation in the case of KLF5, Erbin, and TCFL5 siRNAs. We suggest that KLF5, Erbin, and TCFL5 are essential for MCTS formation and play a key role in the development of tumor diseases.
Asunto(s)
Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica/genética , Esferoides Celulares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proliferación Celular , Neoplasias del Colon/patología , Células HT29 , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/genéticaRESUMEN
In vitro differentiation of embryonic stem (ES) cells results in generation of tissue-specific somatic cells and may represent a powerful tool for general understanding of cellular differentiation and development in vivo. Culturing of most ES cell lines requires murine embryonic fibroblasts (MEF), which may influence adventitiously the genetic differentiation program of ES cells. We compared the expression profile of key developmental genes in the MEF-independent CGR8 ES cell line and in the MEF-dependent D3 ES cell line. Using neomycin-resistant MEFs we demonstrated that MEFs are able to contaminate the D3 ES cells even after removing the MEFs. Subsequently, optimal differentiation conditions were established for the differentiation of CGR8 ES cells into various germ layer cells. Detailed gene expression studies in differentiating CGR8 cells were done by RT-PCR analysis and by microarray analysis demonstrating a general trend of the assessed genes to be expressed either in 3 days- or 10-days old embryoid bodies (EBs) when compared to undifferentiated ES cells. Subsets within the various functional gene classes were defined that are specifically up- or down-regulated in concert. Interestingly, the present results demonstrate that developmental processes toward germ layer formation are irreversible and mostly independent of the culture conditions. Notably, apoptotic and mitochondrial ribosomal genes were down- and up-regulated in 10-days old EBs, respectively, whereas compared to the 3-days old EBs whereas the activity of the extracellular signal-regulated kinase (ERK) 1/2 decreased with progressive development. This article defines a platform for ES cell differentiation and gene expression studies.
Asunto(s)
Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Madre/fisiología , Animales , Apoptosis/genética , Blastocisto/citología , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/genética , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Marcadores Genéticos , Masculino , Proteínas de la Membrana/genética , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Animales , Familia de Multigenes , Factor 3 de Transcripción de Unión a Octámeros/genética , Fosforilación , Células Madre/citología , Factor de Crecimiento Transformador beta/genéticaRESUMEN
BACKGROUND: Members of a class of bHLH transcription factors, namely the hairy (h), Enhancer of split (E(spl)) and hairy-related with YRPW motif (hey) (h/E(spl)/hey) genes are involved in vertebrate somitogenesis and some of them show cycling expression. By sequence comparison, identified orthologues of cycling somitogenesis genes from higher vertebrates do not show an appropriate expression pattern in zebrafish. The zebrafish genomic sequence is not available yet but the genome of Fugu rubripes was recently published. To allow comparative analysis, the currently known Her proteins from zebrafish were used to screen the genomic sequence database of Fugu rubripes. RESULTS: 20 h/E(spl)/hey-related genes were identified in Fugu, which is twice the number of corresponding zebrafish genes known so far. A novel class of c-Hairy proteins was identified in the genomes of Fugu and Tetraodon. A screen of the human genome database with the Fugu proteins yielded 10 h/E(spl)/hey-related genes. By analysing the upstream sequences of the c-hairy class genes in zebrafish, Fugu and Tetraodon highly similar sequence stretches were identified that harbour Suppressor of hairless paired binding sites (SPS). This motif was also discovered in the upstream sequences of the her1 gene in the examined fish species. Here, the Su(h) sites are separated by longer intervening sequences. CONCLUSIONS: Our study indicates that not all her homologues in zebrafish have been isolated. Comparison to the human genome suggests a selective duplication of h/E(spl) genes in pufferfish or loss of members of these genes during evolution to the human lineage.
