(C) Hierarchical clustering analysis of gene expression profiles of three pairwise comparisons (Ad5-siHIF-1alpha group vs. Ad5 group1, Ad5-HIF-1alpha group vs. Ad5 group2, hypoxia group vs. normoxia group). The normalization of all the data of genes with differential expression was handled by clustering analysis using software Gene Spring 7.0. The graph of clustering analysis on the right side is the magnification about the local region (as marked by black border) of the total clustering analysis. Major functional

categories of upregulated genes in response to hypoxia by HIF-1alpha Analysis of genes that were upregulated revealed several large categories of gene products associated with immune response, transport, signal transduction,

cell adhesion/motility, growth factor/cytokines, transcription, inflammatory response, metabolic process, apoptosis and others (Table 1). The gene most highly upregulated by HIF-1alpha was CLIC2. The largest groups upregulated TSA HDAC purchase by HIF-1alpha in NCI-H446 cell were genes associated with transport GW-572016 mouse and the metabolic process. Among the genes associated with transport, the largest category was the SLC (solute carrier) gene family including SLC6A2, SLC9A2, SLC38A6, SLC16A6, SLC41A2, SLC12A8, SLC12A6, SLC39A8 and SLCO4A1. The genes of the SLC family such as SLC2A14 and SLC2A3 and the AKR1 (aldo-keto PF-3084014 solubility dmso reductase 1) family such as AKR1C1, AKR1C2, AKR1C3 and AKR1B10 were associated with the metabolic processes of tumor cells. Ten genes were identified that encode cytokines Sirolimus concentration and growth factors including the known target genes of HIF-1alpha such as VEGF, IGFBP5, PDGFC and CRLF1. Novel upregulated genes that might be implicated as target genes of HIF-1alpha including TNFAIP6, HMOX1, HMGA2, HEY1, PLA2G4A and SOCS1. Another large category

of target genes encoded transcription factors; among these CREM and ZNF277 were target genes of HIF-1alpha. Among the genes encoding inflammatory response factors, 8 genes (TNFAIP6, IL1R1, BDKRB1, C4A, PTGS2, TNFRSF11B, FN1 and IL6) were upregulated. No gene encoding for inflammatory response factors were downregulated by HIF-1alpha. To validate the microarray data, aliquots from the same RNA preparations were analyzed by quantitative real-time PCR for six genes: IGFBP5, IRS4, TNFAIP6, SOCS1, IL-6, VEGF-A. The results of the real-time PCR showed a similar trend of regulation as the microarray data despite the different upregulational fold (Figure 2A). Table 1 65 genes upregulated by HIF-1alpha more than 2.0-fold in three pairwise comparisons UniGeneID Gene name Gene Symbol Fold change(ratio ≥ 2)       Ad5-HIF-1alpha/Ad5 Ad5-siHIF-1alpha/Ad5 Hypoxia/normoxia Immune response Hs.351812 C-type lectin domain family 4, member C CLEC4C 12.99 -9.66 17.54 Hs.190622 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 DDX58 5.28 -3.12 4.77 Hs.163173 interferon induced with helicase C domain 1 IFIH1 3.73 -2.07 4.15 Hs.529053 complement component 3 C3 2.29 -2.10 3.17 Transport Hs.