ABSTRACT
PURPOSE: A goal of this study was to identify and investigate previously unrecognized components of the remodeling process in the progression to heart failure by comparing protein expression in ischemic failing (F) and nonfailing (NF) human hearts. EXPERIMENTAL DESIGN: Protein expression differences were investigated using multidimensional protein identification and validated by Western analysis. This approach detected basal lamina (BL) remodeling, and further studies analyzed samples for evidence of structural BL remodeling. A rat model of pressure overload (PO) was studied to determine whether nonischemic stressors also produce BL remodeling and impact cellular adhesion. RESULTS: Differential protein expression of collagen IV, laminin α2, and nidogen-1 indicated BL remodeling develops in F versus NF hearts Periodic disruption of cardiac myocyte BL accompanied this process in F, but not NF heart. The rat PO myocardium also developed BL remodeling and compromised myocyte adhesion compared to sham controls. CONCLUSIONS AND CLINICAL RELEVANCE: Differential protein expression and evidence of structural and functional BL alterations develop during heart failure. The compromised adhesion associated with this remodeling indicates a high potential for dysfunctional cellular integrity and tethering in failing myocytes. Therapeutically targeting BL remodeling could slow or prevent the progression of heart disease.
Subject(s)
Basement Membrane/metabolism , Collagen Type IV/genetics , Heart Failure/diagnosis , Laminin/genetics , Membrane Glycoproteins/genetics , Myocardial Ischemia/diagnosis , Aged , Animals , Basement Membrane/pathology , Collagen Type IV/metabolism , Disease Models, Animal , Gene Expression Profiling , Gene Expression Regulation , Heart Failure/genetics , Heart Failure/metabolism , Heart Failure/pathology , Humans , Laminin/metabolism , Membrane Glycoproteins/metabolism , Middle Aged , Myocardial Ischemia/genetics , Myocardial Ischemia/metabolism , Myocardial Ischemia/pathology , Myocardium/metabolism , Myocardium/pathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Primary Cell Culture , Rats , Rats, Sprague-DawleyABSTRACT
Steady-state gene expression is a coordination of synthesis and decay of RNA through epigenetic regulation, transcription factors, micro RNAs (miRNAs), and RNA-binding proteins. Here, we present bromouride labeling and sequencing (Bru-Seq) and bromouridine pulse-chase and sequencing (BruChase-Seq) to assess genome-wide changes to RNA synthesis and stability in human fibroblasts at homeostasis and after exposure to the proinflammatory tumor necrosis factor (TNF). The inflammatory response in human cells involves rapid and dramatic changes in gene expression, and the Bru-Seq and BruChase-Seq techniques revealed a coordinated and complex regulation of gene expression both at the transcriptional and posttranscriptional levels. The combinatory analysis of both RNA synthesis and stability using Bru-Seq and BruChase-Seq allows for a much deeper understanding of mechanisms of gene regulation than afforded by the analysis of steady-state total RNA and should be useful in many biological settings.
Subject(s)
Inflammation/genetics , Inflammation/metabolism , RNA Stability , RNA/biosynthesis , RNA/genetics , Bromodeoxyuridine/metabolism , Cell Line , Fibroblasts/metabolism , Gene Expression Regulation , Genome, Human , Humans , Inflammation/etiology , Introns , RNA/metabolism , RNA Processing, Post-Transcriptional , RNA, Mitochondrial , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Transcriptome , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
BACKGROUND: Activating KRAS mutations are important for cancer initiation and progression; and have recently been shown to cause primary resistance to therapies targeting the epidermal growth factor receptor. Therefore, strategies are currently in development to overcome treatment resistance due to oncogenic KRAS. The hypoxia-inducible factors-1α and -2α (HIF-1α and HIF-2α) are activated in cancer due to dysregulated ras signaling. METHODS: To understand the individual and combined roles of HIF-1α and HIF-2α in cancer metabolism and oncogenic KRAS signaling, we used targeted homologous recombination to disrupt the oncogenic KRAS, HIF-1α, and HIF-2α gene loci in HCT116 colon cancer cells to generate isogenic HCT116WT KRAS, HCT116HIF-1α-/-, HCT116HIF-2α-/-, and HCT116HIF-1α-/-HIF-2α-/- cell lines. RESULTS: Global gene expression analyses of these cell lines reveal that HIF-1α and HIF-2α work together to modulate cancer metabolism and regulate genes signature overlapping with oncogenic KRAS. Cancer cells with disruption of both HIF-1α and HIF-2α or oncogenic KRAS showed decreased aerobic respiration and ATP production, with increased ROS generation. CONCLUSION: Our findings suggest novel strategies for treating tumors with oncogenic KRAS mutations.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Colonic Neoplasms/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mitochondria/metabolism , Proto-Oncogene Proteins/metabolism , ras Proteins/metabolism , Adenosine Triphosphate/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Blotting, Western , Cell Line, Tumor , Colonic Neoplasms/genetics , Gene Expression Profiling , HCT116 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mutagenesis, Site-Directed , Oxygen Consumption/genetics , Oxygen Consumption/physiology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , ras Proteins/geneticsABSTRACT
Gene amplification is an important mechanism of oncogene activation in breast and other cancers. Characterization of amplified regions of the genome in breast cancer has led to the identification of important oncogenes including erbB-2/HER-2, C-MYC, and fibroblast growth factor receptor (FGFR) 2. Chromosome 8p11-p12 is amplified in 10-15% of human breast cancers. The putative oncogene FGFR1 localizes to this region; however, we show evidence that FGFR inhibition fails to slow growth of three breast cancer cell lines with 8p11-p12 amplification. We present a detailed analysis of this amplicon in three human breast cancer cell lines using comparative genomic hybridization, traditional Southern and Northern analysis, and chromosome 8 cDNA microarray expression profiling. This study has identified new candidate oncogenes within the 8p11-p12 region, supporting the hypothesis that genes other than FGFR1 may contribute to oncogenesis in breast cancers with proximal 8p amplification.
Subject(s)
Breast Neoplasms/genetics , Chromosomes, Human, Pair 8/genetics , Blotting, Southern , Chromosome Mapping , DNA, Complementary/genetics , Female , Gene Amplification , Gene Expression Regulation, Neoplastic , Genetic Markers , Humans , Oligonucleotide Array Sequence Analysis , Oncogenes , Receptor Protein-Tyrosine Kinases/genetics , Receptor, Fibroblast Growth Factor, Type 1 , Receptors, Fibroblast Growth Factor/genetics , Tumor Cells, CulturedABSTRACT
Cytogenetic and molecular studies have suggested that deletion or rearrangement of sequences that map to the short arm of chromosome 8 may be permissive for tumorigenesis in several organ systems, and in human prostate tumors in particular. In this study, we hypothesized that genes deleted for one copy and localized to the 8p chromosomal region may be transcriptionally down-regulated or ablated in affected human prostate tumor tissues. To test this hypothesis, we used cDNA microarray analysis to determine the transcriptional profiles for 259 transcribed sequences mapping to the 8p chromosomal region for seven human prostate tumor xenografts, completely characterized for numerical and structural alterations on chromosome 8, and five normal human prostate tissues. These experiments identified 33 genes differentially expressed between normal and malignant prostate tissues, the majority of which (28/33, 85%) were transcriptionally down-regulated in malignant compared to normal human prostate tissues. These findings, that haploinsufficiency and transcriptional down-regulation for genes mapping to 8p are largely coincident in human prostate tumors, should provide a powerful tool for the identification of tumor-suppressor genes associated with human prostate cancer initiation and progression.
