A Cell line p53 mutation Type UM-SCC 1 wt UM-SCC5 Exon 5, 157 GTC --> TTC Missense mutation by transversion (Valine --> Phenylalanine UM-SCC6 wt UM-SCC9 wt UM-SCC11A wt UM-SCC11B Exon 7, 242 TGC --> TCC Missense mutation by transversion (Cysteine --> Serine) UM-SCC22A Exon 6, 220 TAT --> TGT Missense mutation by transition (Tyrosine --> Cysteine) UM-SCC22B Exon 6, 220 TAT --> TGT Missense mutation by transition (Tyrosine --> Cysteine) UM-SCC38 Exon 5, 132 AAG --> AAT Missense mutation by transversion (Lysine --> Asparagine) UM-SCC46 Exon 8, 278 CCT --> CGT Missense mutation by transversion (Proline --> Alanine) B 1 Supplementary Methods Cell Lines and Cell Culture A panel of ten established HNSCC cell lines from the University of Michigan series (UM-SCC) was obtained from Dr. T. E. Carey at the University of Michigan, Ann Arbor, MI. The UM-SCC cell lines were derived from eight patients with SCC of the upper aerodigestive tract (supplemental Table 1). Patient age at tumor diagnosis ranged from 37 to 72 years. The cell lines selected were obtained from patients with stage I-IV tumors, distributed among oral, pharyngeal and laryngeal sites. All the patients had aggressive disease, with early recurrence and death within two years of therapy. Cell lines established from single isolates of a patient specimen are designated by a numeric designation, and where isolates from two time points or anatomical sites were obtained, the designation includes an alphabetical suffix (i.e., "A" or "B"). The cell lines were maintained in Eagle's minimal essential media supplemented with 10% fetal bovine serum and penicillin/streptomycin. Human normal keratinocytes (HKC) were obtained from four individuals (Cascade Biologics Inc., Portland, OR), and were maintained in keratinocyte serum-free medium 154CF containing 0.08mM of calcium chloride and supplemented with human keratinocyte growth supplements (HKGS). The final concentration of HKGS in the complete medium are: 0.2% (v/v) of bovine pituitary extract (BPE), 5 μg/ml of bovine insulin, 0.18 μg/ml of hydrocortisone and 5 μg/ml of transferrin, and 0.2ng/ml of human recombinant EGF. All HKC were used within five passages. The cultures were incubated o in a humidified cell culture incubator at 37 C and 5 % of CO2. 1 RNA isolation, Labeling of cDNA, Microarray Hybridization and Image Collection Total RNA was isolated from cultured UM-SCC cells and primary keratinocytes at their log growth phase using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol and stored at -80°C. To make fluorescence-labeled target cDNA by reverse transcription, 50-100 µg total RNA was incubated in a cocktail containing Cy3 or Cy5-dUTP (Amersham Pharmacia Biotech Inc, Piscataway, NJ) and SuperScript II RT (Gibco BRL Technologies, Inc, Gaithersburg, MD). Labeled targets were purified using a Microcon column (Millipore, Bedford, MA). The appropriate Cy3 and Cy5 targets were combined, along with 2 µl (20 µg) mouse COT-1 DNA, 1 µl (8-10 µg) polyA, 2.6 µl 20X SSC and 0.45 µl 10% SDS in a final volume of 15 µl. After denaturation, labeled targets were added to processed 24K human array chips developed by National Human Genome Research Institute (NHGRI, Bethesda, MD), which were then placed in moisturized chambers and incubated overnight (10-16 hours) @ 65oC. The next day, slides were washed for 1 minute in 1X SSC, 1 minute in 0.2X SSC, 10 seconds in 0.05X SSC, then spin dried. Each hybridization condition was repeated at least three times. Fluorescence images were captured by GenePix 4000 microarray scanner (Axon Instruments, Union City, CA) with GenePix Pro software from the same company. The PMT voltage for both channels was adjusted between 750V to 890V range to give same overall intensity between both channels and the raw image was saved in TIFF format. Data Collection, Filtering and Quality Control The 24K cDNA microarray chips containing a total of 23220 spots, which includes known genes, ESTs, hypothetical genes and control spots were obtained from National 2 Human Genome Research Institute (Bethesda, MD). The study was focused on the 12270 known gene set in the array. Raw images were analyzed using the ArraySuite 2.1 extensions (Chen et al., 1997) in the IPLab program (Scanalytics, Inc., Fairfax, VA) to calibrate relative ratios and develop confidence intervals for their significance (Chen et al., 1997). Fluorescence intensities for both dyes (Cy3 and Cy5) and local background subtracted values for individual spots were obtained. Spots were filtered based on RQuality score provided in the software and visually checked. The software extracts information regarding spot quality and assigns an RQuality score to each ratio measurement, with 0 as the lowest measurement quality and 1 as the highest measurement quality. Any spots with less than 0.5 were excluded. For each spot, the calibrated ratio (the median of the pixel-by-pixel ratios of pixel intensities that have the median background intensity subtracted) was used in subsequent analysis. Expression outliners were determined using +3.0 SD and a 99% confidence interval as cutoff. The resulting gene list was further filtered to exclude EST and hypothetical clones and contain expression value from at least three human normal keratinocyte and seven tumor cell lines, resulting in a total of 9273 genes. Data Normalization, Calculation and Statistical Analysis PCA analysis was performed on normalized data from Partek Pro 5.1 software (Partek Inc., St. Louis, MO). Hierarchical clustering was carried out on gene list where the average ratio difference of ten tumor cell lines to normal keratinocytes were more than 2- fold and had a t-test score at P < 0.01. The resulting 969 genes were analyzed by cluster software based on clustering algorithm of Eisen et al (Eisen et al., 1998), and the 3 expression maps of clustered genes were visualized using Java Treeview software (Saldanha et al., 2004). Hierarchical agglomerative clustering was performed by BRB- ArrayTools developed by Dr. Richard Simon and Amy Peng (available at http://linus.nci.nih.gov/BRB-ArrayTools.html). It was applied to the normalized log ratios by using both compact linkage and average linkage and both Euclidean and one minus Pearson correlation distance metrics. Normalized log ratios were median-centered within each gene for all of the cluster analyses. The clustering results obtained by using compact linkage with one minus Pearson correlation distance applied to the 969 probe elements appeared by visual inspection to yield the most distinctive clusters, where attention is focused on the genes responsible for segregating members in sub_A and sub_B by PCA analysis in Figure 1. The presence of significant clustering was also assessed by applying the global test of clustering proposed by McShane et al. to confirm the findings (McShane et al., 2002). Gene Ontology analysis was carried out by Database for Annotation, Visualization, and Integrated Discovery (DAVID); a web-based, client/server application that allows users to access a relational database of functional annotation (NIAID, NIH, Bethesda, MD). Functional annotations are derived primarily from LocusLink at the National Center for Biotechnology Information (NCBI). Annotation pedigrees are provided via direct links to the primary sources of annotation, which also provide additional gene specific information. To analyze the differential expression between the tumor and normal group uniquely, statistical analysis of the data from the hierarchical clustering was performed using BRB-Array Tools, where a class comparison tool was used. Univariate F-tests (BRB-Array Tools 2.0) were performed on these in the two 4 classes. This tool also computes a global permutation test that excludes genes that differ significantly due to chance alone. Genes that showed significant differences (P <0.001) after 2000 permutations were designated as differentially expressed genes. A mixed-model-based F test was used to analyze differential gene expression among three groups of cells, HKC, the sub-group 1 or 2 of UM-SCC cells (Tempelman, 2005; Wolfinger et al., 2001). Restricted maximum likelihood (REML) was used as the estimation method. The F test was performed in mixed model using SAS 9.1 program (SAS Institute Inc. Cary, NC). P < 0.05 was designated as significant difference between two groups. Real Time RT-PCR Gene expression profiles generated from microarray were confirmed by real time quantitative RT-PCR. cDNA synthesis using total RNA was performed by using the High-Capacity cDNA Archive Kit, and Real time quantitative RT-PCR was performed using the Assays-on-Demand™ Gene Expression kits for specific genes and an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocols. PCR was performed together with endogenous control using eukaryotic 18S ribosomal RNA (18S). The amplification was carried out by addition of 30 ng of cDNA in 30 μl of PCR reaction mix (1.5 μl of 20X assay mix and 15 μl of 2X TaqMan Universal Master Mix). Amplification conditions were as follows: activation of enzymes for 2 min at 50oC and 10 min at 95oC, followed by 40 cycles at 15 sec at 95oC and 1 min at 60oC. Relative quantitation of the expression was calculated by normalizing the target gene signals with the 18S endogenous control. An arbitrary unit was calculated after setting CT equaling to 40 as undetectable 5 expression level and used for normalization. In Situ Hybridization Human larynx tumors and normal mucosa were obtained from the Cooperative Human Tissue Network. Specimens were sectioned at 10 µm, mounted onto Poly-L-Lysine microscope slides (Polysciences, Inc, Warrington, PA), and the UM-SCC 11A xenograft tumors were used as a positive control (Ricker et al., 2004). The cRNA probes were synthesized from the following cDNA clones: BAG2 (659 base pairs, Invitrogen); CCNB2 (1315 base pairs, Open Biosystems, Huntsville, AL); PCNA (154 base pairs); and CCND1 (560 base pairs, from Dr.