ABSTRACT: We hypothesized that broad-scale expression profiling would provide insight into the regulatory pathways that control gene expression in response to stress, and potentially identify novel heat-responsive genes. HEp2 cells were heated at 37 to 43 °C for 60 min to gauge the heat shock response, using as a proxy inducible HSP-70 quantified by western blot analysis. Based on these results, microarray experiments were conducted at 37, 40, 41, 42 and 43°C (3 replicates/temperature x 5 groups = 15 U95Aver2 GeneChips). Using linear modeling, we compared the sets of microarrays at 40, 41, 42 and 43°C with the 37°C baseline temperature and took the union of the genes exhibiting differential gene expression signal to create two sets of âheat shock responseâ genes, each set reflecting either increased or decreased RNA abundance. Leveraging human and mouse orthologous alignments, we used the two lists of co-expressed genes to predict transcription factor binding sites in silico, including those for heat shock factor 1 (HSF1) and heat shock factor 2 (HSF2) transcription factors. We discovered HSF1 and HSF2 binding sites in 15 genes not previously associated with the heat shock response. We conclude that microarray experiments coupled with upstream promoter analysis can be used to identify novel genes that respond to heat shock. Additional experiments are required to validate these putative heat shock proteins and facilitate a deeper understanding of the mechanisms involved during the stress response. Experiment Overall Design: Cell Culture: HEp2 cells were acquired from American Type Culture Cell (Rockville, MD) and cultured in EMEM supplemented with 10% fetal bovine serum (Gibco Laboratories, Grand Island, NY), 1% nonessential amino acids (Gibco), and 1.5g/L sodium bicarbonate. The cell cultures were maintained at 37°C in a humidified incubator in an atmosphere of 5% CO2 â 95% air and passaged at a 1:3 ratio with trypsin every 5-7 days. Experiment Overall Design: Heat shock and RNA extraction: After growing to confluency, cells were heated for 60 min in an incubator at 37, 38, 39, 40, 41, 42, or 43°C as measured by a sterile probe placed into each plateâs media. After heating, media was removed and ice cold PBS was immediately added and cells were scraped from plates and put into 1.5mL centrifuge tubes and pelletted. PBS was removed and replaced with 1 mL of TRIzol® and vortexed vigorously (Invitrogen, Carlsbad, CA) for 5min. RNA was then extracted as per manufactures protocol. RNA integrity was assessed by gel electrophoresis and the Agilent Bioanalyzer 2100 machine (Agilent Technologies, Waldbronn, Germany). Experiment Overall Design: Affymetrix cRNA target generation: RNA purification (RNAeasy, Qiagen), cDNA synthesis (Invitrogen, Carlsbad, CA) and biotinylated cRNA (Invitrogen, Carlsbad, CA) were all performed in accordance with manufacturerâs instructions (Affymetrix, Inc., Santa Clara, CA). Fragmented target cRNA was hybridized with U95v2 GeneChip using Affymetrix® equipment at the Alvin J. Siteman Cancer Center Genechip Facility. Fluorescent signal was collected using an Affmetrix  G2500A GeneArray Scanner (Hewlett-Packard, Inc., Cupertino, CA). Experiment Overall Design: GeneChip microarray data were generated at cellular temperatures of 37, 40, 41, 42, and 43°C (U95Av2 GeneChips®, ~10,000 genes per microarray; 3 technical replicates per temperature = 15 GeneChips®).
