Cell Culture

Human coronary artery endothelial cells (ECs), smooth muscle cells (SMCs), human mammalian epithelial cells (HMECs), and renal proximal tubule epithelial cells (RPTECs) were purchased from Cambrex ( East Rutherford, New Jersey, United States), and cultured in specified medium as suggested by manufacturer. To create hypoxic conditions, 70%–80% confluent cells were placed in a tissue culture incubator with 2% O2 and 5% CO2 at 37 °C. To create anoxic conditions, 70%–80% confluent cells placed in an anaerobic chamber (Sheldon, Cornelius, Oregon, United States) with less than 0.02% O2 and 5% CO2 at 37 °C.

HIF-1a Quantitative Real-Time PCR

Total RNA was purified with Absolutely RNA microprep kit (Stratagene, La Jolla, California, United States). Quantitative real-time PCR was performed on an ABI PRISM 7900H machine (Applied Biosystems, Foster City, California, United States) with TaqMan reagents (Applied Biosystems). Primer sequences used for HIF-1a were: 5′ primer, 5′-CTCACCCAACGAAAAATTACAGAA-3′; 3′ primer, 5′-ATTGAGTGCAGGGTCAGCACTAC-3′, and probe, FAM-CATTACCCACCGCTGAAACGCCAA-TAMRA. TaqMan β-actin detection reagents (Applied Biosystems) were used as an internal control for quantitation. Quantitative real-time PCR was performed at 95 °C for 10 min followed by 40 cycles of denaturing at 95 °C for 15 s and annealing/extending at 60 °C for 1 min.

HIF-1a Immunoblotting

Cell lysates were prepared by adding RIPA buffer immediately after hypoxia or anoxia. Total protein was quantitated with the BCA Protein Assay Reagent Kit (Pierce, Rockford, Illinois, United States) and equal amounts of protein loaded in each lane of a 10% SDS-PAGE gel followed by transfer to a PVDF membrane (Bio-Rad, Hercules, California, United States) at 90 V for 2 h. After blocking with 5% nonfat milk for 1 h at room temperature, the membrane was incubated with monoclonal antibodies specific for HIF-1a (BD Biosciences, San Jose, California, United States) overnight at 4 °C. After washing, horseradish peroxidase-linked anti-mouse IgG (Amersham Biosciences, Piscataway, New Jersey, United States) was used as a secondary antibody, and incubated with the membrane for 45 min at room temperature. Signal was detected by ECL Western blotting detection reagents (Amersham Biosciences).

HIF-1 a Dicer RNA Interference

The following three sets of primers were used to generate fragments for HIF-1a Dicer RNA interference (RNAi) and incorporated T7 promoter sequences: HIF-1a forward primer-1, 5′-GCGTAATACGACTCACTATAGGGACACTGGTGGCTCACTACC-3′; HIF-1a reverse primer-1, 5′-GCGTAATACGACTCACTATAGGGTCCAGGTTTAACAATTTCATAGGCC-3′; HIF-1a forward primer-2, 5′-GCGTAATACGACTCACTATAGGGATGGTTCTCACAGATGATGGTGAC-3′; HIF-1a reverse primer-2, 5′-GCGTAATACGACTCACTATAGGGTTGAGCGGCCTAAAAGTTCTTC-3′; HIF-1a forward primer-3, 5′-GCGTAATACGACTCACTATAGGGCCAAGAATTCTCAACCACAGTGC-3′; and HIF-1a reverse primer-3, 5′-GCGTAATACGACTCACTATAGGGTCGGAAGGACTAGGTGTCTGATC-3′.

Firefly luciferase (GL3) and Renilla luciferase (RL) were used as controls. Sequences used were designed based upon previous reports [1]. DNA fragments with T7 promoters were generated by PCR and subjected to in vitro transcription to produce dsRNAs (550 bp) using the MEGAscript in vitro transcription kit (Ambion, Austin, Texas, United States). dsRNAs were treated with DNase I, and incubated with r-Dicer at 37 °C in 250 mM NaCl, 30 mM HEPES (pH 8.0), 0.05 mM EDTA, 2.5 mM MgCl 2 overnight. Dicer-treated small interfering RNA (d-siRNA) was purified using the Micro-to-Midi Total RNA Purification System (Invitrogen) and transfected into RPTECs by electroporation by using the Basic Nucleofector Kit for primary mammalian epithelial cells and Nucleofector Device according to the directions provided by the manufacturer (Amaxa Biosystems, Gaithersburg, Maryland, United States). 24 h after transfection, the cells were subjected to hypoxia and RNAs harvested 12 h later for analysis.

cDNA Microarrays and Hybridization

We used human cDNA microarrays containing 42,000 elements that represent 27291 unique genes. Arrays were produced at the Stanford Functional Genomic Facility. The RNAs were purified by FastTrack (Invitrogen), and fluorescently labeled cDNAs were hybridized to the array in a two-color comparative format, with the experimental samples labeled with one fluorophore (Cy-5) and a reference pool of mRNA labeled with a second fluorophore (Cy-3) [2].

Data Filtering and Analysis

Array images were scanned by using an Axon Scanner 4000B (Axon Instruments, Union City, California, United States), and image analysis was performed by using Genepix Pro version 3.0.6.89 (Axon Instruments). Data were expressed as the log2 ratio of fluorescence intensities of the sample and the reference, for each element on the array [2]. Data were filtered to exclude elements that did not have at least a 2.5-fold intensity/background ratio in at least 60% of the arrays. The time course data from each cell type under hypoxia (2%) and anoxia (0%) were normalized to the ambient air control harvested at the same time, and the subset of elements that varied from the baseline by at least 3-fold in at least four samples was selected for further analysis. The data were hierarchically clustered by using the cluster program [3], and displayed by using TreeView. The changes in gene expression for each gene were evaluated at each time point through zero transformation [4] by subtracting log2(red/green) of the normoxia (~21% O2) measurement from the corresponding log2(red/green) ratio at hypoxia (2% O2) or anoxia (0% O2) at the same time points. The resulting values represent log2(red hypoxia/red normoxia), or log2(red anoxia/red normoxia) and so on, and indicate changes associated with either hypoxia (2% O2) or anoxia (0% O2). To identify genes with changes only one of the four cell types, we used multi-class significance analysis of microarrays (SAM) [5] to analyze variations of gene expression after zero-transformation associated with ECs, SMCs, HMECs or RPTECs. The cell type-specific gene list was selected to have a false discovery rate (FDR) of 3.7 %, using 100 iterations. For detailed procedures and complete data, please see Figure S1, Tables S1 and S2.

Analysis of an Epithelial Hypoxia Signature in Human Cancers

The epithelial hypoxia signature gene list consists of 253 unique image clones on the cDNA Stanford array by selecting a gene cluster showing induction in the tested epithelial cells (HMECs and RPTECs). For gene expression analysis of renal cell carcinoma [6], breast cancers [7], and ovarian cancer, the expression value of these clones was extracted and genes were selected for further analysis for which the corresponding array elements had fluorescent hybridization signals at least 2.5-fold greater than the local background fluorescence. We further restricted our analysis to genes for which adequate data were obtained in at least 80% of experiments. The image clones in the epithelial hypoxia signature that satisfied all the above criteria were used to stratify tumors in different datasets based on their hypoxia response with hierarchical clustering. For the analysis of breast cancer samples of Netherlands Cancer Institute (NKI), 35 of 253 unique image clones could not be mapped to a Unigene cluster. The 218 remaining clones were mapped to 168 unique Unigene clusters. The 168 Unigene cluster represented 180 unique sequences on the Rosetta/NKI oligo array. Cross-checking gene names revealed 22 probes that could not confidently be contributed to genes in the original hypoxia signature. These were removed, resulting in 158 matched probes. These 158 were the matching probes to 123 unique Unigene clusters. In order to overcome possible overestimation of Unigene clones that were matched to more than one probe on the NKI array, the probes that were not uniquely match to one Unigene cluster were averaged. Genes were mean centered and clustered and visualized in TreeView. Based on genes highly expressed in the hypoxia response, two groups of patients were identified. These were called, respectively, high- and low-hypoxia response. Overall survival was defined by death from any cause. Distant metastasis-free survival was defined by a distant metastasis as a first recurrence event; data on all patients were censored on the date of the last follow-up visit, death from causes other than breast cancer, the recurrence of local or regional disease, or the development of a second primary cancer, including contralateral breast cancer.

Kaplan-Meier survival curves were compared by the Cox-Mantel log-rank test in Winstat for Excel (R. Fitch Software, Germany). Multivariate analysis by the Cox proportional hazard method was performed using the software package SPSS 11.5 (SPSS, Chicago, Illinois, United States).

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2. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, et al. (2000) Molecular portraits of human breast tumours. Nature 406: 747-752.

3. Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A 95: 14863-14868.

4. Baldwin DN, Vanchinathan V, Brown PO, Theriot JA (2003) A gene-expression program reflecting the innate immune response of cultured intestinal epithelial cells to infection by Listeria monocytogenes. Genome Biol 4: R2.

5. Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98: 5116-5121.

6. Higgins JP, Shinghal R, Gill H, Reese JH, Terris M, et al. (2003) Gene expression patterns in renal cell carcinoma assessed by complementary DNA microarray. Am J Pathol 162: 925-932.

7. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, et al. (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 98: 10869-10874.