ABSTRACT
The effects of hypoxia on gene transcription are mainly mediated by a transcription factor complex termed hypoxia-inducible factor (HIF). Genetic manipulation of animals and studies of humans with rare hereditary disease have shown that modifying the HIF pathway affects systems-level physiological responses to hypoxia. It is, however, an open question whether variations in systems-level responses to hypoxia between individuals could arise from variations within the HIF system. This study sought to determine whether variations in the responsiveness of the HIF system at the cellular level could be detected between normal individuals. Peripheral blood lymphocytes (PBL) were isolated on three separate occasions from each of 10 healthy volunteers. After exposure of PBL to eight different oxygen tensions ranging from 20% to 0.1%, the expression levels of four HIF-regulated transcripts involved in different biological pathways were measured. The profile of expression of all four transcripts in PBL was related to oxygen tension in a curvilinear manner. Double logarithmic transformation of these data resulted in a linear relationship that allowed the response to be parameterized through a gradient and intercept. Analysis of variance (ANOVA) on these parameters showed that the level of between-subject variation in the gradients of the responses that was common across all four HIF-regulated transcripts was significant (P = 0.008). We conclude that statistically significant variation within the cellular response to hypoxia can be detected between normal humans. The common nature of the variability across all four HIF-regulated genes suggests that the source of this variation resides within the HIF system itself.
Subject(s)
Hypoxia/genetics , Lymphocytes/metabolism , Oxygen/metabolism , Transcription, Genetic , Adrenomedullin/genetics , Adrenomedullin/metabolism , Adult , Analysis of Variance , Cells, Cultured , Female , Fructose-Bisphosphate Aldolase/genetics , Fructose-Bisphosphate Aldolase/metabolism , Humans , Hypoxia/blood , Hypoxia/metabolism , Linear Models , Male , Phenotype , Procollagen-Proline Dioxygenase/genetics , Procollagen-Proline Dioxygenase/metabolism , RNA, Messenger/metabolism , Up-Regulation , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Young AdultABSTRACT
Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-alpha subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-alpha subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1alpha and HIF-2alpha, with HIF-1alpha suppression manifesting substantially greater effects than HIF-2alpha suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.
Subject(s)
Cell Hypoxia/genetics , Cell Hypoxia/physiology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Ketoglutarate Dehydrogenase Complex/antagonists & inhibitors , Transcription Factors/metabolism , Amino Acids, Dicarboxylic/pharmacology , Basic Helix-Loop-Helix Transcription Factors , Cell Line , Enzyme Inhibitors/pharmacology , Gene Expression Profiling , Gene Expression Regulation/drug effects , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Transcription Factors/antagonists & inhibitors , Transcription Factors/geneticsABSTRACT
Real competitive PCR (rcPCR) has been shown to have high sensitivity, reproducibility, and high-throughput potential. We describe further development and evaluation of this methodology as a tool for measuring nucleic acid abundance within a cell. Modifications to the original protocol allow analysis of gene expression levels using standard conditions regardless of mRNA abundance and assay type, thereby increasing throughput and ease of reaction setup while decreasing optimization time. In addition, we have developed a software package, TITAN, to automatically analyze the results. The details are relevant to researchers performing competitive PCR using any detection technique. The effectiveness of the described developments is demonstrated using 12 genes known to have differential expression in cell lines grown under normal and hypoxic conditions. Quantitative and qualitative comparisons to real-time PCR are presented. It is also demonstrated that the technique is capable of detecting submicroscopic chromosomal DNA deletions.
