ABSTRACT
Tumor necrosis factor alpha (TNF alpha) is a proinflammatory cytokine with important roles in regulating inflammatory responses as well as cell cycle proliferation and apoptosis. Although TNFalpha stimulates apoptosis, it also activates the transcription factor NF-kappa B, and studies have shown that inhibition of NF-kappa B potentiates the cytotoxicity of TNFalpha. Since several chemotherapy agents act like TNFalpha to both promote apoptosis and activate NF-kappa B, understanding the role of NF-kappa B in suppressing apoptosis may have significant clinical applications. To understand the effects of stimulation with TNFalpha and the role of NF-kappa B in regulating this response, a 23k human cDNA microarray was used to screen TNFalpha-inducible genes in HeLa cells. Real-time PCR verified expression changes in 16 of these genes and revealed three distinct temporal patterns of expression after TNFalpha stimulation. Using RNA interference to disrupt expression of the p65 subunit of NF-kappa B, all but two of the genes were shown to depend on this transcription factor for their expression, which correlated well with the existence of NF-kappa B binding sites in most of their promoters. Inflammatory, proapoptotic, and antiapoptotic genes were all shown to be regulated by NF-kappa B, demonstrating the wide variety of targets activated by NF-kappa B signaling and the necessity of differentiating among these genes for therapeutic purposes.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation , NF-kappa B/physiology , RNA Interference , Computer Systems , Gene Expression Regulation/drug effects , HeLa Cells/drug effects , HeLa Cells/metabolism , Humans , NF-kappa B/genetics , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Promoter Regions, Genetic/genetics , Transcription Factor RelA , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
PURPOSE: The purpose of this study was to profile gene expression changes in colorectal tumors to identify new targets and strategies for the management of this disease. EXPERIMENTAL DESIGN: cDNA microarray analysis was used to detect differences in gene expression between normal tissue and colon tumors and polyps isolated from 20 patients. To identify genes that are important in regulating the growth properties of colorectal cancer, RNA interference (RNAi) was used to disrupt expression of several of the overexpressed genes in a colon tumor cell line, HCT116, which showed similar patterns of gene expression as many of the patient tumors. RESULTS: Expression changes of > or =2-fold in approximately one-third of the patients were consistently observed for 2632 of a total of 9592 genes (574 up-regulated genes and 2058 down-regulated genes). Subsequent analysis of 13 genes by quantitative real-time PCR confirmed the reliability of this analysis. RNAi-mediated disruption of the expression of one of these genes, survivin, a potent inhibitor of apoptosis, severely reduced tumor growth both in vitro and in an in vivo xenograft model. CONCLUSIONS: The combined use of microarray analysis and RNAi provides an excellent system to define the role of specific genes that are up-regulated in cancer lead to the increased in vitro and in vivo growth of colon tumors.
Subject(s)
Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , RNA Interference , Animals , Blotting, Western , Cell Cycle , Cell Division , DNA, Complementary/metabolism , Down-Regulation , Female , Humans , Inhibitor of Apoptosis Proteins , Male , Mice , Microtubule-Associated Proteins/biosynthesis , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/physiology , Neoplasm Proteins , Neoplasm Transplantation , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Oligonucleotides/chemistry , Proto-Oncogene Proteins c-myc/physiology , RNA/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Survivin , Time Factors , Transfection , Tumor Cells, Cultured , Up-RegulationABSTRACT
ARROGANT (ARRay OrGANizing Tool) is a software tool developed to facilitate the identification, annotation and comparison of large collections of genes or clones. The objective is to enable users to compile gene/clone collections from different databases, allowing them to design experiments and analyze the collections as well as associated experimental data efficiently. ARROGANT can relate different sequence identifiers to their common reference sequence using the UniGene database, allowing for the comparison of data from two different microarray experiments. ARROGANT has been successfully used to analyze microarray expression data for colon cancer, to compile genes potentially related to cardiac diseases for subsequent resequencing (to identify single nucleotide polymorphisms, SNPs), to design a new comprehensive human cDNA microarray for cancer, to combine and compare expression data generated by different microarrays and to provide annotation for genes on custom and Affymetrix chips.
