Cellular and transcriptomic analysis of NS0 cell response during exposure to hypoxia

Mammalian cells have the ability to alter their gene expression in order to survive or adapt to a variety of environment stresses including hypoxic stress. Maintaining oxygen supply has been accepted as essential for cell survival and growth. To determine the cellular and molecular changes which take place under oxygen deprivation, an NS0 cell line producing a human-mouse chimeric antibody was cultured under hypoxic conditions (<1%). Various cellular parameters such as viability, productivity, metabolism, apoptosis and cell cycle were studied and notable changes were shown to be accompanied by changes in metabolic rates. When the cells where exposed to hypoxia for 48 h, cell growth was suppressed and cell death was detected. To better understand and explore the mechanisms underpinning these biological alterations and to identify the genes involved in the genetic reprogramming, genome-wide analyses were performed using GeneChip Mouse Genome arrays. The gene expression profiling generated by the microarray technique revealed that hypoxia, even in the early stages (12h), induces significant changes in gene expression in NS0 cells. The primary responses to hypoxia within the cells were: (1) the up-regulation of pathways such as glycolysis that ultimately lead to alternative routes of ATP generation and increased oxygen availability; and (2) the down-regulation of genes involved in purine/pyrimidine and one carbon pool metabolisms required for DNA and RNA synthesis. By combining gene expression and physiological changes under hypoxia, it was possible to explore the mechanisms of hypoxia-induced alterations in more depth.     

Glycosylation and post-translational modification gene expression analysis by DNA microarrays for cultured mammalian cells

DNA microarray analysis of gene expression has become a valuable tool for bioprocessing research aimed at improving therapeutic protein yields. The highly parallel nature of DNA microarray technology allows researchers to assess hundreds of gene simultaneously, essentially enabling genome-wide snapshots. The quality and amount of therapeutic proteins produced by cultured mammalian cells rely heavily on the culture environment. In order to implement beneficial changes to the culture environment, a better understanding of the relationship between the product quality and culture environment must be developed. By analyzing gene expression levels under various environmental conditions, light can be shed on the underlying mechanisms. This paper describes a method for evaluating gene expression changes for cultured NS0 cells, a mouse-derived myeloma cell line, under culture environment conditions, such as ammonia buildup, known to affect product quality. These procedures can be easily adapted to other environmental conditions and any mammalian cell lines cultured in suspension, so long as a sufficient number of gene sequences are publicly available.     

Cellular and molecular analysis of plasmodium development in Physarum.

The development of an amoeba into a plasmodium involves extensive changes in cellular organisation and gene expression. The genetic basis of a number of recessive mutations that block plasmodium development has been elucidated. The stage at which development becomes abnormal has been determined for all the mutants, as has the terminal phenotype. In order to investigate the changes in gene expression that accompany plasmodium development, a cDNA library has been made using RNA isolated from cell populations in which development was occurring.     

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.     

A novel network profiling analysis reveals system changes in epithelial-mesenchymal transition

Patient-specific analysis of molecular networks is a promising strategy for making individual risk predictions and treatment decisions in cancer therapy. Although systems biology allows the gene network of a cell to be reconstructed from clinical gene expression data, traditional methods, such as bayesian networks, only provide an averaged network for all samples. Therefore, these methods cannot reveal patient-specific differences in molecular networks during cancer progression. In this study, we developed a novel statistical method called NetworkProfiler, which infers patient-specific gene regulatory networks for a specific clinical characteristic, such as cancer progression, from gene expression data of cancer patients. We applied NetworkProfiler to microarray gene expression data from 762 cancer cell lines and extracted the system changes that were related to the epithelial-mesenchymal transition (EMT). Out of 1732 possible regulators of E-cadherin, a cell adhesion molecule that modulates the EMT, NetworkProfiler, identified 25 candidate regulators, of which about half have been experimentally verified in the literature. In addition, we used NetworkProfiler to predict EMT-dependent master regulators that enhanced cell adhesion, migration, invasion, and metastasis. In order to further evaluate the performance of NetworkProfiler, we selected Krueppel-like factor 5 (KLF5) from a list of the remaining candidate regulators of E-cadherin and conducted in vitro validation experiments. As a result, we found that knockdown of KLF5 by siRNA significantly decreased E-cadherin expression and induced morphological changes characteristic of EMT. In addition, in vitro experiments of a novel candidate EMT-related microRNA, miR-100, confirmed the involvement of miR-100 in several EMT-related aspects, which was consistent with the predictions obtained by NetworkProfiler.     

Gene expression analysis

The response of cells to extracellular signals usually requires altered expression of many genes, possibly including several distinct metabolic pathways. In some cases, only a subset of genes involved in such responses are known, which requires techniques to analyze changes in the expression of multiple genes, both known and unknown. Three techniques, two-dimensional gel electrophoresis, differential display, and gene discovery arrays, provide opportunities for measuring changes in gene expression levels, as well as for identifying novel gene products.     

Genomewide analysis of gene expression associated with Tcof1 in mouse neuroblastoma

Mutations in the Treacher Collins syndrome gene, TCOF1, cause a disorder of craniofacial development. We manipulated the levels of Tcof1 and its protein treacle in a murine neuroblastoma cell line to identify downstream changes in gene expression using a microarray platform. We identified a set of genes that have similar expression with Tcof1 as well as a set of genes that are negatively correlated with Tcof1 expression. We also showed that the level of Tcof1 and treacle expression is downregulated during differentiation of neuroblastoma cells into neuronal cells. Inhibition of Tcof1 expression by siRNA induced morphological changes in neuroblastoma cells that mimic differentiation. Thus, expression of Tcof1 and treacle synthesis play an important role in the proliferation of neuroblastoma cells and we have identified genes that may be important in this pathway.     

Wrestling with pleiotropy: genomic and topological analysis of the yeast gene expression network

The vast majority (>95%) of single-gene mutations in yeast affect not only the expression of the mutant gene, but also the expression of many other genes. These data suggest the presence of a previously uncharacterized "gene expression network"--a set of interactions between genes which dictate gene expression in the native cell environment. Here, we quantitatively analyze the gene expression network revealed by microarray expression data from 273 different yeast gene deletion mutants.(1) We find that gene expression interactions form a robust, error-tolerant "scale-free" network, similar to metabolic pathways(2) and artificial networks such as power grids and the internet.(3-5) Because the connectivity between genes in the gene expression network is unevenly distributed, a scale-free organization helps make organisms resistant to the deleterious effects of mutation, and is thus highly adaptive. The existence of a gene expression network poses practical considerations for the study of gene function, since most mutant phenotypes are the result of changes in the expression of many genes. Using principles of scale-free network topology, we propose that fragmenting the gene expression network via "genome-engineering" may be a viable and practical approach to isolating gene function.     

Genome-wide gene expression analysis in cancer cells reveals 3D growth to affect ECM and processes associated with cell adhesion but not DNA repair

Cell morphology determines cell behavior, signal transduction, protein-protein interaction, and responsiveness to external stimuli. In cancer, these functions profoundly contribute to resistance mechanisms to radio- and chemotherapy. With regard to this aspect, this study compared the genome wide gene expression in exponentially growing cell lines from different tumor entities, lung carcinoma and squamous cell carcinoma, under more physiological three-dimensional (3D) versus monolayer cell culture conditions. Whole genome cDNA microarray analysis was accomplished using the Affymetrix HG U133 Plus 2.0 gene chip. Significance analysis of microarray (SAM) and t-test analysis revealed significant changes in gene expression profiles of 3D relative to 2D cell culture conditions. These changes affected the extracellular matrix and were mainly associated with biological processes like tissue development, cell adhesion, immune system and defense response in contrast to terms related to DNA repair, which lacked significant alterations. Selected genes were verified by semi-quantitative RT-PCR and Western blotting. Additionally, we show that 3D growth mediates a significant increase in tumor cell radio- and chemoresistance relative to 2D. Our findings show significant gene expression differences between 3D and 2D cell culture systems and indicate that cellular responsiveness to external stress such as ionizing radiation and chemotherapeutics is essentially influenced by differential expression of genes involved in the regulation of integrin signaling, cell shape and cell-cell contact.